U.S. patent application number 10/227282 was filed with the patent office on 2003-12-04 for screening assays for identifying differentiation-inducing agents and production of differentiated cells for cell therapy.
This patent application is currently assigned to Advanced Cell Technology. Invention is credited to Chapman, Karen, Page, Raymond, Scholer, Hans, West, Michael D..
Application Number | 20030224345 10/227282 |
Document ID | / |
Family ID | 23219468 |
Filed Date | 2003-12-04 |
United States Patent
Application |
20030224345 |
Kind Code |
A1 |
West, Michael D. ; et
al. |
December 4, 2003 |
Screening assays for identifying differentiation-inducing agents
and production of differentiated cells for cell therapy
Abstract
The invention relates to assays for screening growth factors,
adhesion molecules, immunostimulatory molecules, extracellular
matrix components and other materials, alone or in combination,
simultaneously or temporally, for the ability to induce directed
differentiation of pluripotent and multipotent stem cells.
Inventors: |
West, Michael D.;
(Southborough, MA) ; Page, Raymond; (Southbridge,
MA) ; Scholer, Hans; (Kennett Square, PA) ;
Chapman, Karen; (SouthBorough, MA) |
Correspondence
Address: |
Pillsbury Winthrop LLP
Intellectual Property Group
P.O. Box 10500
McLean
VA
22102
US
|
Assignee: |
Advanced Cell Technology
Worcester
MA
|
Family ID: |
23219468 |
Appl. No.: |
10/227282 |
Filed: |
August 26, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60314316 |
Aug 24, 2001 |
|
|
|
Current U.S.
Class: |
435/4 ; 435/350;
435/351; 435/353; 435/354; 435/366 |
Current CPC
Class: |
C12N 2501/998 20130101;
G01N 33/5011 20130101; C12N 2501/115 20130101; C12N 2506/04
20130101; G01N 33/5023 20130101; G01N 33/5073 20130101; C12N 5/0652
20130101; C12N 2501/155 20130101; C12N 2501/23 20130101; C12N 5/069
20130101; C12N 5/0657 20130101; C12N 2506/03 20130101; C12Q
2600/158 20130101; G01N 33/5091 20130101; G01N 2333/475 20130101;
C12N 2506/02 20130101; C12Q 1/6888 20130101; G01N 33/5061 20130101;
C12N 2501/15 20130101; G01N 2333/52 20130101 |
Class at
Publication: |
435/4 ; 435/366;
435/353; 435/354; 435/350; 435/351 |
International
Class: |
C12Q 001/00; C12N
005/06; C12N 005/08 |
Claims
What is claimed:
1. A method for evaluating the differentiation of totipotent,
nearly totipotent, or pluripotent stem cells, or cells therefrom,
in response to one or more chemical or biological agents or
physical conditions, comprising: (a) separating individual
totipotent, nearly totipotent, or pluripotent stem cells, or cells
therefrom, or groups of such cells, in culture medium into one or a
plurality of separate wells which may be open or closed, which
wells may be in the same or different apparatus; (b) exposing said
separate wells of cells to one or more putative
differentiation-inducing conditions simultaneously or sequentially;
and (c) screening said individual cells or groups of cells to
detect markers of differentiation of said individual cells or
groups of cells.
2. The method of claim 1, wherein said totipotent, nearly
totipotent, or pluripotent stem cells are selected from the group
consisting of inner cell mass (ICM) cells, embryonic stem (ES)
cells, embryonic germ (EG) cells, embryos consisting of one to
about 400 cells, embryoid body cells, morula-derived cells,
embryonic pluripotent cells, and cells therefrom.
3. The method of claim 1, wherein said nearly totipotent, or
pluripotent stem cells, or cells therefrom, are selected from the
group consisting of human cells, primate cells, bovine cells,
porcine cells, murine cells, rat cells, sheep cells, canine and
feline cells.
4. The method of claim 1, wherein said one or more putative
differentiation-inducing conditions are selected from the group
consisting of growth factors, cytokines, tissue extracts, nucleic
acids, factors involved in cell-to-cell interactions, adhesion
molecules and extracellular matrix components, extracts of
extracellular components from tissue, media components,
environmental conditions, and living cells that induce
differentiation by cell-cell interactions.
5. The method of claim 4, wherein said growth factors, chemokines,
and cytokines are selected from the group consisting of the
Fibroblast Growth Factor family of proteins (FGF1-23) including but
not limited to FGF basic (146 aa), FGF basic (157 AA), FGF acidic,
the TGF beta family of proteins including but not limited to
TGF-beta 1, TGF-beta 2, TGF-beta sRII, Latent TGF-beta, the Tumor
necrosis factor (TNF) superfamily (TNFSF) including but not limited
to TNFSF1-18, including TNF-alpha, TNF-beta, the insulin-like
growth factor family incuding but not limited to IGF-1 and their
binding proteins including but not limited to IGFBP-1, II-1 R rp2,
IGFBP-5, IGFBP-6, the matrix metalloproteinases including but not
limited to MMP-1, CF, MMP-2, CF, MMP-2 (NSA-expressed), CF, MMP-7,
MMp-8, MMP-10, MMP-9, TIMP-1, CF, TIMP-2 and other growth factors
and cytokines including but not limited to PDGF, Flt-3 ligand, Fas
Ligand, B7-1 (CD80), B7-2(CD86), DR6, IL-13 R alpha, IL-15 R alpha,
GRO beta/CXCL2 (aa 39-107), IL 1-18, II-8/CXCL8, GDNF, G-CSF,
GM-CSF, M-GSF, PDGF-BB, PDGF-AA, PDGF-AB, IL-2 sR alpha, IL-2 sR
beta, Soluble TNF RII, IL-6 sR, Soluble gp130, PD-ECGF, IL-4 sR,
beta-ECGF, TGF-alpha, TGF-beta sRII, TGF-beta 5, LAP (TGF-beta 1),
BDNF, LIF sR alpha, LIF, KGF/FGF-7, Pleiotrophin, ENA-78/CXCL5,
SCF, beta-NGF, CNTF, Midkine, HB-EGF, SLPI, Betacellulin,
Amphiregulin, PIGF, Angiogenin, IP-10/CXCL10, NT-3, NT-4, MIP-1
alpha/CCL3, MIP-1 beta/CCL4, I-309/CCL1, GRO alpha/CXCL1, GRO
beta/CXCL2, GRO gamma/CXCL3, Rantes/CCL5, MCP-1/CCL2, MCP-2/CCL8,
MCP-3/CCL7, IFN-gamma, Erythropoietin, Thrombopoietin, MIF, IGF-I,
IGF-II, VEGF, HGF, Oncostatin M, HRG-alpha (EGF Domain), TGF-beta
2, CNTF R alpha, Tie-2/Fc Chimera, BMP-4, BMPR-IA, Eotaxin/CCL11,
VEGF R1 (Fit-1), PDGF sR alpha, HCC-1/CCL14, CTLA-4, MCP-4/CCL13,
GCP-2/CXCL6, TECK/CCL25, MDC/CCL22, Activin A,
Eotaxin-2/MPIF-2/CCL24, Eotaxin-3/CCL-26 (aa 24-94), TRAIL R1
(DR4), VEGF R3 (Fit-4)/SDF-1 alpha(PBSF)/CXCL12, MSP, BMP-2,
HVEM/VEGF R2 (KDR), Ephrin-A3, MIP-3 alpha/CCL20, MIP-3 beta/CCL19,
Fractalkine/CX3CL1 (Chemokine Domain), TARC/CCL17, 6Ckine/CCL21,
p75 Neurotrophin R (NGF R), SMDF, Neurturin, Leptin R/Fc Chimera,
MIG/CXCL9, NAP-2/CXCL7, PARC/CCL18, Cardiotrophin-1 (CT-1), GFR
alpha-2, BMP-5, IL-8/CXCL8 (Endothelial Cell Derived), Tie-1, Viral
CMV UL146, VEGF-D, Angiopoietin-2, Inhibin A, TRANCE/RANK L, CD6/Fc
Chimera, CF, dMIP-1 delta/LKN-1/CCL15(68 aa), TRAIL R3/Fc Chimera,
Soluble TNF RI, Activin RIA, EphA1, E-Cadherin, ENA-70, ENA-74,
Eotaxin-3/CCL26, ALCAM, FGFR1 alpha (IIIc), Activin B, FGFT1 beta
(IIIc), LIGHT, FGFR2 beta(IIIb), DNAM-1, Follistatin, GFR alpha-3,
gp 130, I-TAC/CXCL11, IFN-gamma R1, IGFBP-2, IGFBP-3, Inhibin B,
Prolactin CF, RANK, FGFR2 beta (IIIc), FGFR4, TrkB, GITR, MSP R,
GITR Ligand, Lymphotactin/XCL1, FGFR2 alpha (IIIc), Activin AB,
ICAM-3 (CD50), ICAM-1 (CD54), TNF RII, L-Selectin (CD62L,
BLC/BCA-1/CXCL13, HCC-4/CCL16, ICAM-2 (CD102), IGFBP-4,
Osteoprotegerin)OPG), uPAR, Activin RIB, VCAM-1 (CD106), CF,
BMPR-II, IL-18 R, IL-12 R beta 1, Dtk, LBP, SDF-1 alpha
(PBSF)/CXCL12 (synthetic), E-Selectin (CD62E), L-Selectin (CD62L),
P-Selectin (CD62P), ICAM-1 (CD54), VCAM-1 (CD106), CD31 (PECAM-1),
hedgehog family of proteins, Interleukin-10, Epidermal Growth
Factor, Heregulin, HER4, Heparin Binding Epidermal Growth Factor,
bFGF, MIP-18, MIP-2, MCP-1, MCP-5, NGF, NGF-B, leptin, Interferon
A, Interferon A/D, Interferon B, Interferon Inducible Protein-10,
Insulin Like Growth Factor-II, IGBFBP/IGF-1 Complex, C10, Cytokine
Induced Neutrophil Chemoattractant 2, Cytokine Induced Neutrophil
Chemoattractant 2B, Cytokine Induced Neutrophil Chemoattractant 1,
Cytokine Responsive Gene-2, and any fragment thereof and their
neutralizing antibodies.
6. The method of claim 4, wherein said factors involved in
cell-cell interactions are selected from the group consisting of
the ADAM (A Disintegrin and Metalloproteinase) family of proteins
including ADAM 1, 2, 3A, 3B, 4-31 and TS1-9, ADAMTSs (ADAMs with
thrombospondin motifs), Reprolysins, metzincins, zincins, and zinc
metalloproteinases and their neutralizing antibodies.
7. The method of claim 4, wherein said adhesion molecules are
selected from the group consisting of Ig superfamily CAM's,
Integrins, Cadherins and Selectins and their neutralizing
antibodies.
8. The method of claim 4, wherein said nucleic acids that may be
tested include but are not limited to those that encode or block by
antisense, ribozyme activity, or RNA interference with
transcription factors that are involved in regulating gene
expression during differentiation, genes for growth factors,
cytokines, and extracellular matrix components, or other molecular
activities that regulate differentiation.
9. The method of claim 4 wherein said cell-cell interactions
include placing the cells being assayed in cell-cell contact with
cells of another differentiated cell type, or in the presence of
media conditioned by cells of another differentiated cell type.
10. The method of claim 4 wherein said tissue extracts include but
are not limited to materials derived from early stage embryos,
fetuses, or adult tissues that may be tested include but are not
limited to acellular extracellular matrix prepared by the detergent
extraction of tissue from embryoid bodies, primitive endoderm,
mesoderm, and ectoderm, and the anlagen of differentiating organs
and tissues or living cells or tissues that when co-cultured with
the subject cells cause an induction of differentiation.
11. The method of claim 4 wherein said environmental conditions
include but are not limited to oxygen tension, carbon dioxide
tension, nitric oxide tension, temperature, pH, mechanical stress,
altered culture substrates such as two vs. three dimensional
substrates, growth on beads, inside cylinders, or porous
substrates.
12. The method of claim 4, wherein said extracellular matrix
components are selected from the group including but are not
limited to Keratin Sulphate Proteoglycan, Laminin, Chondroitin
Sulphate A, SPARC, beta amyloid precursor protein, beta amyloid,
presenilin 1,2, apolipoprotein E, thrombospondin-1,2, Heparan
Sulphate, Heparan sulphate proteoglycan, Matrigel, Aggregan,
Biglycan, Poly-L-Ornithine, the collagen family including but not
limited to Collagen I-IV, Poly-D-Lysine, Ecistatin (Viper Venom),
Flavoridin (Viper Venom), Kistrin (Viper Venom), Vitronectin,
Superfibronectin, Fibronectin Adhesion-Promoting peptide,
Fibronectin Fragment III-C, Fibronectin Fragment-30 KDA,
Fibronectin-Like Polymer, Fibronectin Fragment 45 KDA, Fibronectin
Fragment 70 KDA, Asialoganglioside-GM, Disialoganglioside-GOLA,
Monosialo Ganglioside-GM.sub.1, Monosialoganglioside-GM.sub.2,
Monosialoganglioside-GM.sub.3,, Methylcellulose, Keratin Sulphate
Proteoglycam, Laminin and Chondroitin Sulphate A.
13. The method of claim 1, wherein said individual cells or
individual groups of cells are separated into separate wells of one
or more multi-well plates.
14. The method of claim 1, further comprising: isolating primary
and/or progenitor cells from reference tissues and placing said
primary and/or progenitor cells into separate vessels of a
microarray thereby forming a control reference library; and after
exposing said separate vessels of totipotent, nearly totipotent, or
pluripotent stem cells, or cells therefrom, to said one or more
putative differentiation-inducing compounds either simultaneously
or sequentially; comparing said individual totipotent, nearly
totipotent, or pluripotent stem cells, or cells therefrom, or
groups of cells, to said reference library in order to evaluate the
differentiation of said individual cells or groups of cells.
15. The method of claim 14, wherein said reference library of
primary cells is constructed in one or more multi-well plates.
16. The method of claim 14, wherein said primary and/or progenitor
cells for said reference library include one or more of brain
cells, heart cells, liver cells, skin cells, pancreatic cells,
blood cells, reproductive cells, nerve cells, sensory cells,
vascular cells, skeletal cells, immune cells, lung cells, muscle
cells, and kidney cells.
17. The method of claim 14, wherein said totipotent, nearly
totipotent, or pluripotent stem cells, or cells therefrom, and said
reference library cells are compared using functional assays
specific for the particular primary cells in the reference
library.
18. The method of claim 17, wherein said functional assays measure
the production of enzymes or metabolites produced by said reference
primary and/or progenitor cells.
19. The method of claim 14, wherein said exposed totipotent, nearly
totipotent, or pluripotent stem cells, or cells therefrom, and said
reference library cells are compared by testing for the presence of
specific cell surface markers or receptors.
20. The method of claim 19, wherein the presence of said cell
surface markers is detected using labeled antibodies or ligands
specific for said cell surface markers or receptors.
21. A method for evaluating the differentiation of a totipotent,
nearly totipotent, or pluripotent stem cell, or cells therefrom, or
a group of such cells, in response to different compounds or
combinations of compounds, comprising: (a) separating individual
totipotent, nearly totipotent, or pluripotent stem cells, or cells
therefrom, or groups of such cells, into one or a plurality of
separate vessels which may be open or closed, which vessels may be
in the same or different apparatus; (b) exposing said separate
vessels of cells to a panel of different putative
differentiation-inducing compounds or combinations thereof
simultaneously or sequentially; and (c) comparing said individual
cells or groups of cells to a reference panel of differentiated or
partially differentiated cells in order to evaluate the
differentiation of said individual totipotent, nearly totipotent,
or pluripotent stem cells, or cells therefrom, or groups of such
cells.
22. The method of claim 21, wherein said totipotent, nearly
totipotent, or pluripotent stem cells, are selected from the group
consisting of inner cell mass (ICM) cells, embryonic stem (ES)
cells, embryonic germ (EG) cells, embryos consisting of one to
about 400 cells, embryoid body cells, morula-derived cells,
embryonic pluripotent cells, and cells therefrom.
23. The method of claim 21, wherein said individual totipotent,
nearly totipotent, or pluripotent stem cells, or cells therefrom,
are selected from the group consisting of human cells, primate
cells, bovine cells, porcine cells, murine cells, rat cells, sheep
cells, and rabbit cells.
24. The method of claim 21, wherein said one or more putative
differentiation-inducing compounds are selected from the group
consisting of growth factors, cytokines, factors involved in
cell-to-cell interactions, adhesion molecules and extracellular
matrix components.
25. The method of claim 24, wherein said growth factors and
cytokines are selected from the group consisting of TGF-beta 2,
PDGF, Fas Ligand, FGF acidic, B7-1 (CD80), B7-2(CD86), DR6, IL-13 R
alpha, IL-15 R alpha, GRO beta/CXCL2 (aa 39-107), IL 1-18,
II-8/CXCL8, TNF-alpha, TNF-beta, GDNF, G-CSF, GM-CSF, M-GSF,
PDGF-BB, PDGF-M, PDGF-AB, IL-2 sR alpha, IL-2 sR beta, Soluble TNF
RII, IL-6 sR, Soluble gp130, PD-ECGF, IL-4 sR, beta-ECGF, FGF basic
(146 aa), FGF basic (157 AA), FGF1-21, TGF-alpha, TGF-beta 1,
TGF-beta sRII, TGF-beta 5, LAP (TGF-beta 1), BDNF, LIF sR alpha,
LIF, KGF/FGF-7, Pleiotrophin, ENA-78/CXCL5, SCF, beta-NGF, CNTF,
Midkine, HB-EGF, SLPI, Betacellulin, Amphiregulin, PIGF,
Angiogenin, IP-10/CXCL10, NT-3, NT-4, MIP-1 alpha/CCL3, MIP-1
beta/CCL4, I-309/CCL1, GRO alpha/CXCL1, GRO beta/CXCL2, GRO
gamma/CXCL3, Rantes/CCL5, MCP-1/CCL2, MCP-2/CCL8, MCP-3/CCL7,
IFN-gamma, Erythropoietin, Thrombopoietin, MIF, IGF-I, IGF-II,
VEGF, HGF, Oncostatin M, HRG-alpha (EGF Domain), Latent TGF-beta,
TGF-beta 2, CNTF R alpha, Tie-2/Fc Chimera, BMP-4, BMPR-IA,
Eotaxin/CCL11, VEGF R1 (Fit-1), PDGF sR alpha, HCC-1/CCL14, CTLA-4,
MCP-4/CCL13, GCP-2/CXCL6, TECK/CCL25, MDC/CCL22, Activin A,
TGF-beta sRII, Eotaxin-2/MPIF-2/CCL24, Eotaxin-3/CCL-26 (aa 24-94),
TRAIL R1 (DR4), VEGF R3 (Fit-4)/SDF-1 alpha(PBSF)/CXCL12, MSP,
BMP-2, HVEM/VEGF R2 (KDR), Ephrin-A3, MIP-3 alpha/CCL20, MIP-3
beta/CCL19, Fractalkine/CX3CL1 (Chemokine Domain), TARC/CCL17,
6Ckine/CCL21, p75 Neurotrophin R (NGF R), SMDF, Neurturin, Leptin
R/Fc Chimera, MIG/CXCL9, NAP-2/CXCL7, PARC/CCL18, Cardiotrophin-1
(CT-1), GFR alpha-2, BMP-5, IL-8/CXCL8 (Endothelial Cell Derived),
Tie-1, Viral CMV UL146, VEGF-D, Angiopoietin-2, Inhibin A,
TRANCE/RANK L, CD6/Fc Chimera, CF, dMIP-1 delta/LKN-1/CCL15(68 aa),
TRAIL R3/Fc Chimera, Soluble TNF RI, Activin RIA, EphA1,
E-Cadherin, ENA-70, ENA-74, Eotaxin-3/CCL26, ALCAM, FGFR1 alpha
(IIIc), Activin B, FGFT1 beta (IIIc), LIGHT, FGFR2 beta(IIIb),
DNAM-1, Follistatin, GFR alpha-3, gp 130, I-TAC/CXCL11, IFN-gamma
R1, IGFBP-2, IGFBP-3, Inhibin B, Prolactin CF, RANK, FGFR2 beta
(IIIc), FGFR4, TrkB, GITR, MSP R, GITR Ligand, Lymphotactin/XCL1,
FGFR2 alpha (IIIc), Activin AB, ICAM-3 (CD50), ICAM-1 (CD54), TNF
RII, L-Selectin (CD62L, BLC/BCA-1/CXCL13, HCC-4/CCL16, ICAM-2
(CD102), IGFBP-4, Osteoprotegerin)OPG), uPAR, Activin RIB, VCAM-1
(CD106), CF, BMPR-II, IL-18 R, IL-12 R beta 1, Dtk, LBP, IGFBP-1,
II-1 R rp2, IGFBP-5, IGFBP-6, MMP-1, CF, MMP-2, CF, MMP-2
(NSA-expressed), CF, MMP-7, MMp-8, MMP-10, CF, MMP-9<CF, TIMP-1,
CF, TIMP-2, CF, SDF-1 alpha (PBSF)/CXCL12 (synthetic), E-Selectin
(CD62E), L-Selectin (CD62L), P-Selectin (CD62P), ICAM-1 (CD54),
VCAM-1 (CD106), CD31 (PECAM-1).
26. The method of claim 24, wherein said factors involved in
cell-cell interactions are selected from the group consisting of
ADAM 1, 2, 3A, 3B, 4-31, TS1-9.
27. The method of claim 24, wherein said adhesion molecules are
selected from the group consisting of Ig superfamily CAM's,
Integrins, Cadherins and Selectins.
28. The method of claim 24, wherein said extracellular matrix
components are selected from the group consisting of Keratin
Sulphate Proteoglycan, Laminin, Chondroitin Sulphate A,
Thrombospondin-1, Heparan Sulphate, Aggregan, Biglycan,
Poly-L-Ornithine, Collagen I, Collagen II, Collagen IV,
Poly-D-Lysine, Ecistatin (Viper Venom), Flavoridin (Viper Venom),
Kistrin (Viper Venom), Vitronectin, Superfibronectin, Fibronectin
Adhesion-Promoting peptide, Fibronectin Fragment Ill-C, Fibronectin
Fragment-30 KDA, Fibronectin-Like Polymer, Fibronectin Fragment 45
KDA, Fibronectin Fragment 70 KDA, Prostaglandin F.sub.2,
Somatostatin, Thyrotropin Releasing Hormone, L-Thyroxine,
3,3,5-Triiodo-L-Thyronine, L-Ascorbic Acid, Asialoganglioside-GM,
Disialoganglioside-GOLA, Monosialo Ganglioside-GM.sub.1,
Monosialoganglioside-GM.sub.2, Monosialoganglioside-GM.sub.3,
Lipids, Transferrin, B-Cyclodextrin, Ascorbate, Fetuin, Heparin,
2-Mercaptoethanol, Horse Serum, DMSO, Chicken Serum, Goat Serum,
Rabbit Serum, Human Serum, MIP-18, MIP-2, MCP-1, MCP-5, NGF, NGF-B,
Pituitary Extract, Stromal Cell Factor, Conditioned Medium,
Hybridoma Medium, d-Aldosterone, Dexamethasone, DHT, B-Estradiol,
Glucagon, Insulin, Progesterone, Prostaglandin-D.sub.2,
Prostaglandin-E.sub.1, Prostaglandin-E.sub.2,
Prostaglandin-F.sub.2, Serum-Free Medium, Gene Therapy Medium,
MDBK-GM Medium, QBSF-S1, Endothelial Medium, Keratinocyte Medium,
Melanocyte Medium, Interleukin-10, Epidermal Growth Factor, Heparin
Binding Epidermal Growth Factor, bFGF, Gly-His-Lys, Insulin Like
Growth Factor-II, IGBFBP/IGF-1 Complex, C10, Cytokine Induced
Neutrophil Chemoattractant 2, Cytokine Induced Neutrophil
Chemoattractant 2B, Cytokine Induced Neutrophil Chemoattractant 1,
Cytokine Responsive Gene-2, Endothelial Cell Growth Supplement,
Interferon A, Interferon A/D, Interferon B, Interferon Inducible
Protein-10, Leptin, Methylcellulose, Keratin Sulphate Proteoglycam,
Laminin and Chondroitin Sulphate A.
29. The method of claim 21, wherein said individual totipotent,
nearly totipotent, or pluripotent stem cells, or cells therefrom,
or individual groups of such cells are separated into separate
wells of one or more multi-well plates.
30. The method of claim 21, wherein said reference differentiated
or partially differentiated cell is a primary or progenitor cell
selected from the group consisting of brain cells, heart cells,
liver cells, skin cells, pancreatic cells, blood cells,
reproductive cells, nerve cells, sensory cells, vascular cells,
skeletal cells, immune cells, lung cells, muscle cells, and kidney
cells.
31. The method of claim 21, wherein said exposed totipotent, nearly
totipotent, or pluripotent stem cells, or cells therefrom, and said
reference cell are compared using functional assays specific for
the reference cell.
32. The method of claim 31, wherein said functional assay measures
the production of one or more enzymes or metabolites produced by
said reference cell.
33. The method of claim 21, wherein said exposed totipotent, nearly
totipotent, or pluripotent stem cells, or cells therefrom, and said
reference cell are compared by testing for the presence of specific
cell surface markers or receptors.
34. The method of claim 33, wherein the presence of said cell
surface markers is detected using labeled antibodies or ligands
specific for said cell surface markers or receptors.
35. The method of claim 21, wherein said exposed totipotent, nearly
totipotent, or pluripotent stem cells, or cells therefrom, and said
reference cell are compared by comparing RNA expression
profiles.
36. A method for evaluating the differentiation of a totipotent,
nearly totipotent, or pluripotent stem cell, or cells therefrom, or
a group of such cells, in response to different compounds or
combinations of compounds, comprising: (a) isolating a transfected
totipotent, nearly totipotent, or pluripotent stem cell, or cells
therefrom, wherein said cell is transfected with at least one
reporter gene, the expression of which is operably linked to a
promoter that is activated when the cell is induced to
differentiate or partially differentiate; (b) expanding said
transfected cell in culture; (c) separating individual transfected
cells or individual groups of cells into one or a plurality of
separate vessels which may be open or closed, which vessels may be
in the same or different apparatus; (d) systematically exposing
said separate vessels of transfected cells simultaneously or
sequentially to a panel of different putative
differentiation-inducing compounds or combinations thereof; and (e)
analyzing said individual transfected cells or groups of cells in
order to detect expression of said at least one reporter gene.
37. The method of claim 35, wherein said totipotent, nearly
totipotent, or pluripotent stem cells, are selected from the group
consisting of inner cell mass (ICM) cells, embryonic stem (ES)
cells, embryonic germ (EG) cells, embryos consisting of one to
about 400 cells, embryoid body cells, morula-derived cells,
embryonic pluripotent cells, and cells therefrom.
38. The method of claim 35, wherein said totipotent, nearly
totipotent, or pluripotent stem cells, or cells therefrom, are
selected from the group consisting of human cells, primate cells,
bovine cells, porcine cells, murine cells, rat cells and sheep
cells.
39. The method of claim 35, wherein said one or more putative
differentiation-inducing compounds are selected from the group
consisting of growth factors, cytokines, factors involved in
cell-to-cell interactions, adhesion molecules and extracellular
matrix components.
40. The method of claim 39, wherein said growth factors and
cytokines are selected from the group consisting of TGF-beta 2,
PDGF, Fas Ligand, FGF acidic, B7-1(CD80), B7-2(CD86), DR6, IL-13 R
alpha, IL-15 R alpha, GRO beta/CXCL2 (aa 39-107), IL 1-18,
II-8/CXCL8, TNF-alpha, TNF-beta, GDNF, G-CSF, GM-CSF, M-GSF,
PDGF-BB, PDGF-AA, PDGF-AB, IL-2 sR alpha, IL-2 sR beta, Soluble TNF
RII, IL-6 sR, Soluble gp130, PD-ECGF, IL-4 sR, beta-ECGF, FGF basic
(146 aa), FGF basic (157 M), FGF1-21, TGF-alpha, TGF-beta 1,
TGF-beta sRII, TGF-beta 5, LAP (TGF-beta 1), BDNF, LIF sR alpha,
LIF, KGF/FGF-7, Pleiotrophin, ENA-78/CXCL5, SCF, beta-NGF, CNTF,
Midkine, HB-EGF, SLPI, Betacellulin, Amphiregulin, PIGF,
Angiogenin, IP-10/CXCL10, NT-3, NT-4, MIP-1 alpha/CCL3, MIP-1
beta/CCL4, I-309/CCL1, GRO alpha/CXCL1, GRO beta/CXCL2, GRO
gamma/CXCL3, Rantes/CCL5, MCP-1/CCL2, MCP-2/CCL8, MCP-3/CCL7,
IFN-gamma, Erythropoietin, Thrombopoietin, MIF, IGF-I, IGF-II,
VEGF, HGF, Oncostatin M, HRG-alpha (EGF Domain), Latent TGF-beta,
TGF-beta 2, CNTF R alpha, Tie-2/Fc Chimera, BMP-4, BMPR-IA,
Eotaxin/CCL11, VEGF R1 (Fit-1), PDGF sR alpha, HCC-1/CCL14, CTLA-4,
MCP-4/CCL13, GCP-2/CXCL6, TECK/CCL25, MDC/CCL22, Activin A,
TGF-beta sRII, Eotaxin-2/MPIF-2/CCL24, Eotaxin-3/CCL-26 (aa 24-94),
TRAIL R1 (DR4), VEGF R3 (Fit-4)/SDF-1 alpha(PBSF)/CXCL12, MSP,
BMP-2, HVEM/VEGF R2 (KDR), Ephrin-A3, MIP-3 alpha/CCL20, MIP-3
beta/CCL19, Fractalkine/CX3CL1 (Chemokine Domain), TARC/CCL17,
6Ckine/CCL21, p75 Neurotrophin R (NGF R), SMDF, Neurturin, Leptin
R/Fc Chimera, MIG/CXCL9, NAP-2/CXCL7, PARC/CCL18, Cardiotrophin-1
(CT-1), GFR alpha-2, BMP-5, IL-8/CXCL8 (Endothelial Cell Derived),
Tie-1, Viral CMV UL146, VEGF-D, Angiopoietin-2, Inhibin A,
TRANCE/RANK L, CD6/Fc Chimera, CF, dMIP-1 delta/LKN-1/CCL15(68 aa),
TRAIL R3/Fc Chimera, Soluble TNF RI, Activin RIA, EphA1,
E-Cadherin, ENA-70, ENA-74, Eotaxin-3/CCL26, ALCAM, FGFR1 alpha
(IIIc), Activin B, FGFT1 beta (IIIc), LIGHT, FGFR2 beta(IIIb),
DNAM-1, Follistatin, GFR alpha-3, gp 130, I-TAC/CXCL11, IFN-gamma
RI, IGFBP-2, IGFBP-3, Inhibin B, Prolactin CF, RANK, FGFR2 beta
(IIIc), FGFR4, TrkB, GITR, MSP R, GITR Ligand, Lymphotactin/XCL1,
FGFR2 alpha (IIIc), Activin AB, ICAM-3 (CD50), ICAM-1 (CD54), TNF
RII, L-Selectin (CD62L, BLC/BCA-1/CXCL13, HCC-4/CCL16, ICAM-2
(CD102), IGFBP-4, Osteoprotegerin)OPG), UPAR, Activin RIB, VCAM-1
(CD106), CF, BMPR-II, IL-18 R, IL-12 R beta 1, Dtk, LBP, IGFBP-1,
II-1 R rp2, IGFBP-5, IGFBP-6, MMP-1, CF, MMP-2, CF, MMP-2
(NSA-expressed), CF, MMP-7, MMp-8, MMP-10, CF, MMP-9<CF, TIMP-1,
CF, TIMP-2, CF, SDF-1 alpha (PBSF)/CXCL12 (synthetic), E-Selectin
(CD62E), L-Selectin (CD62L), P-Selectin (CD62P), ICAM-1 (CD54),
VCAM-1 (CD106), and CD31 (PECAM-1).
41. The method of claim 39, wherein said factors involved in
cell-cell interactions are selected from the group consisting of
ADAM 1, 2, 3A, 3B, 4-31, TS1-9.
42. The method of claim 39, wherein said adhesion molecules are
selected from the group consisting of Ig superfamily CAM's,
Integrins, Cadherins and Selectins.
43. The method of claim 39, wherein said extracellular matrix
components are selected from the group consisting of Keratin
Sulphate Proteoglycan, Laminin, Chondroitin Sulphate A,
Thrombospondin-1, Heparan Sulphate, Aggregan, Biglycan,
Poly-L-Ornithine, Collagen I, Collagen II, Collagen IV,
Poly-D-Lysine, Ecistatin (Viper Venom), Flavoridin (Viper Venom),
Kistrin (Viper Venom), Vitronectin, Superfibronectin, Fibronectin
Adhesion-Promoting peptide, Fibronectin Fragment III-C, Fibronectin
Fragment-30 KDA, Fibronectin-Like Polymer, Fibronectin Fragment 45
KDA, Fibronectin Fragment 70 KDA, Prostaglandin F.sub.2,
Somatostatin, Thyrotropin Releasing Hormone, L-Thyroxine,
3,3,5-Triiodo-L-Thyronine, L-Ascorbic Acid, Asialoganglioside-GM,
Disialoganglioside-GOLA, Monosialo Ganglioside-GM.sub.1,
Monosialoganglioside-GM.sub.2, Monosialoganglioside-GM.sub.3,
Lipids, Transferrin, B-Cyclodextrin, Ascorbate, Fetuin, Heparin,
2-Mercaptoethanol, Horse Serum, DMSO, Chicken Serum, Goat Serum,
Rabbit Serum, Human Serum, MIP-18, MIP-2, MCP-1, MCP-5, NGF, NGF-B,
Pituitary Extract, Stromal Cell Factor, Conditioned Medium,
Hybridoma Medium, d-Aldosterone, Dexamethasone, DHT, B-Estradiol,
Glucagon, Insulin, Progesterone, Prostaglandin-D.sub.2,
Prostaglandin-E1, Prostaglandin-E.sub.2, Prostaglandin-F.sub.2,
Serum-Free Medium, Gene Therapy Medium, MDBK-GM Medium, QBSF-S1,
Endothelial Medium, Keratinocyte Medium, Melanocyte Medium,
Interleukin-10, Epidermal Growth Factor, Heparin Binding Epidermal
Growth Factor, bFGF, Gly-His-Lys, Insulin Like Growth Factor-II,
IGBFBP/IGF-1 Complex, C10, Cytokine Induced Neutrophil
Chemoattractant 2, Cytokine Induced Neutrophil Chemoattractant 2B,
Cytokine Induced Neutrophil Chemoattractant 1, Cytokine Responsive
Gene-2, Endothelial Cell Growth Supplement, Interferon A,
Interferon A/D, Interferon B, Interferon Inducible Protein-10,
Leptin, Methylcellulose, Keratin Sulphate Proteoglycam, Laminin and
Chondroitin Sulphate A.
44. The method of claim 35, wherein said individual transfected
embryonic cells or individual groups of embryonic cells are
separated into separate wells of one or more multi-well plates.
45. The method of claim 35, wherein said promoter is specifically
expressed in primary or progenitor cells selected from the group
consisting of endodermal cells, mesodermal cells, ectodermal cells,
brain cells, heart cells, liver cells, skin cells, pancreatic
cells, blood cells, reproductive cells, nerve cells, sensory cells,
vascular cells, skeletal cells, immune cells, lung cells, muscle
cells, and kidney cells.
46. The method of claim 35, wherein said reporter gene encodes a
polypeptide capable of producing a detectable signal, wherein said
signal is coupled to transcription from said promoter.
47. The method of claim 46, further comprising: (f) quantitatively
determining the amount of detectable signal; and (g) comparing said
amount of detectable signal with the amount of signal produced by
the same number of said transfected embryonic cells in the absence
of any test compound.
48. The method of claim 46, wherein said detectable signal is
detectable by the naked eye or after microscopic, photographic or
radiographic analysis, after contacting said exposed cells with a
reagent selected from the group consisting of chromogenic
substrates, dyes, sugars, antibodies, ligands and primers.
49. The method of claim 46, wherein said polypeptide is selected
from the group consisting of enhanced green fluoresence protein
(EGFP), luciferase, chloramphenical acetyltransferase,
.beta.-glucuronidase, .beta.-galactosidase, neomycin
phosphotransferase, alkaline phosphatase, guanine xanthine
phosphoribosyltransferase and .beta.-lactamase.
50. A method for evaluating the differentiation of a totipotent,
nearly totipotent, or pluripotent stem cells, or cells therefrom,
or a group of such cells in response to one or more compounds,
comprising: (a) obtaining a library of transfected totipotent,
nearly totipotent, or pluripotent stem cells, or cells therefrom,
each transfected with at least one reporter gene, the expression of
which is linked to a promoter that is activated when the cell is
induced to differentiate or partially differentiate; (b) separating
individual members of said library into one or a plurality of
separate vessels which may be open or closed, which vessels may be
in the same or different apparatus; (c) exposing said separate
vessels of transfected cells simultaneously or sequentially to the
same one or more putative differentiation-inducing compounds; and
(d) analyzing said individual members of said library in order to
detect expression of said at least one reporter gene.
51. The method of claim 50, wherein said totipotent, nearly
totipotent, or pluripotent stem cells, are selected from the group
consisting of inner cell mass (ICM) cells, embryonic stem (ES)
cells, embryonic germ (EG) cells, embryos consisting of one to
about 400 cells, embryoid body cells, morula-derived cells,
embryonic pluripotent cells, and cells therefrom.
52. The method of claim 50, wherein said totipotent, nearly
totipotent, or pluripotent stem cells, or cells therefrom, are
selected from the group consisting of human cells, primate cells,
bovine cells, porcine cells, murine cells, rat cells, sheep cells,
and rabbit cells.
53. The method of claim 50, wherein said one or more putative
differentiation-inducing compounds are selected from the group
consisting of growth factors, cytokines, factors involved in
cell-to-cell interactions, adhesion molecules and extracellular
matrix components.
54. The method of claim 53, wherein said growth factors and
cytokines are selected from the group consisting of TGF-beta 2,
PDGF, Fas Ligand, FGF acidic, B7-1(CD80), B7-2(CD86), DR6, IL-13 R
alpha, IL-15 R alpha, GRO beta/CXCL2 (aa 39-107), IL 1-18,
II-8/CXCL8, TNF-alpha, TNF-beta, GDNF, G-CSF, GM-CSF, M-GSF,
PDGF-BB, PDGF-M, PDGF-AB, IL-2 sR alpha, IL-2 sR beta, Soluble TNF
RII, IL-6 sR, Soluble gp130, PD-ECGF, IL-4 sR, beta-ECGF, FGF basic
(146 aa), FGF basic (157 AA), FGF1-21, TGF-alpha, TGF-beta 1,
TGF-beta sRII, TGF-beta 5, LAP (TGF-beta 1), BDNF, LIF sR alpha,
LIF, KGF/FGF-7, Pleiotrophin, ENA-78/CXCL5, SCF, beta-NGF, CNTF,
Midkine, HB-EGF, SLPI, Betacellulin, Amphiregulin, PIGF,
Angiogenin, IP-10/CXCL10, NT-3, NT-4, MIP-1 alpha/CCL3, MIP-1
beta/CCL4, I-309/CCL1, GRO alpha/CXCL1, GRO beta/CXCL2, GRO
gamma/CXCL3, Rantes/CCL5, MCP-1/CCL2, MCP-2/CCL8, MCP-3/CCL7,
IFN-gamma, Erythropoietin, Thrombopoietin, MIF, IGF-I, IGF-II,
VEGF, HGF, Oncostatin M, HRG-alpha (EGF Domain), Latent TGF-beta,
TGF-beta 2, CNTF R alpha, Tie-2/Fc Chimera, BMP-4, BMPR-IA,
Eotaxin/CCL11, VEGF R1 (Fit-1), PDGF sR alpha, HCC-1/CCL14, CTLA-4,
MCP-4/CCL13, GCP-2/CXCL6, TECK/CCL25, MDC/CCL22, Activin A,
TGF-beta sRII, Eotaxin-2/MPIF-2/CCL24, Eotaxin-3/CCL-26 (aa 24-94),
TRAIL R1 (DR4), VEGF R3 (Fit-4)/SDF-1 alpha(PBSF)/CXCL12, MSP,
BMP-2, HVEM/VEGF R2 (KDR), Ephrin-A3, MIP-3 alpha/CCL20, MIP-3
beta/CCL19, Fractalkine/CX3CL1 (Chemokine Domain), TARC/CCL17,
6Ckine/CCL21, p75 Neurotrophin R (NGF R), SMDF, Neurturin, Leptin
R/Fc Chimera, MIG/CXCL9, NAP-2/CXCL7, PARC/CCL18, Cardiotrophin-1
(CT-1), GFR alpha-2, BMP-5, IL-8/CXCL8 (Endothelial Cell Derived),
Tie-1, Viral CMV UL146, VEGF-D, Angiopoietin-2, Inhibin A,
TRANCE/RANK L, CD6/Fc Chimera, CF, dMIP-1 delta/LKN-1/CCL15(68 aa),
TRAIL R3/Fc Chimera, Soluble TNF RI, Activin RIA, EphA1,
E-Cadherin, ENA-70, ENA-74, Eotaxin-3/CCL26, ALCAM, FGFR1 alpha
(IIIc), Activin B, FGFT1 beta (IIIc), LIGHT, FGFR2 beta(IIIb),
DNAM-1, Follistatin, GFR alpha-3, gp 130, I-TAC/CXCL11, IFN-gamma
RI, IGFBP-2, IGFBP-3, Inhibin B, Prolactin CF, RANK, FGFR2 beta
(IIIc), FGFR4, TrkB, GITR, MSP R, GITR Ligand, Lymphotactin/XCL1,
FGFR2 alpha (IIIc), Activin AB, ICAM-3 (CD50), ICAM-1 (CD54), TNF
RII, L-Selectin (CD62L, BLC/BCA-1/CXCL13, HCC-4/CCL16, ICAM-2
(CD102), IGFBP-4, Osteoprotegerin)OPG), uPAR, Activin RIB, VCAM-1
(CD106), CF, BMPR-II, IL-18 R, IL-12 R beta 1, Dtk, LBP, IGFBP-1,
II-1 R rp2, IGFBP-5, IGFBP-6, MMP-1, CF, MMP-2, CF, MMP-2
(NSA-expressed), CF, MMP-7, MMp-8, MMP-10, CF, MMP-9<CF, TIMP-1,
CF, TIMP-2, CF, SDF-1 alpha (PBSF)/CXCL12 (synthetic), E-Selectin
(CD62E), L-Selectin (CD62L), P-Selectin (CD62P), ICAM-1 (CD54),
VCAM-1 (CD106), CD31 (PECAM-1).
55. The method of claim 53, wherein said factors involved in
cell-cell interactions are selected from the group consisting of
ADAM 1, 2, 3A, 3B, 4-31, TS1-9.
56. The method of claim 53, wherein said adhesion molecules are
selected from the group consisting of Ig superfamily CAM's,
Integrins, Cadherins and Selectins.
57. The method of claim 53, wherein said extracellular matrix
components are selected from the group consisting of Keratin
Sulphate Proteoglycan, Laminin, Chondroitin Sulphate A,
Thrombospondin-1, Heparan Sulphate, Aggregan, Biglycan,
Poly-L-Ornithine, Collagen I, Collagen II, Collagen IV,
Poly-D-Lysine, Ecistatin (Viper Venom), Flavoridin (Viper Venom),
Kistrin (Viper Venom), Vitronectin, Superfibronectin, Fibronectin
Adhesion-Promoting peptide, Fibronectin Fragment III-C, Fibronectin
Fragment-30 KDA, Fibronectin-Like Polymer, Fibronectin Fragment 45
KDA, Fibronectin Fragment 70 KDA, Prostaglandin F.sub.2,
Somatostatin, Thyrotropin Releasing Hormone, L-Thyroxine,
3,3,5-Triiodo-L-Thyronine, L-Ascorbic Acid, Asialoganglioside-GM,
Disialoganglioside-GOLA, Monosialo Ganglioside-GM.sub.1,
Monosialoganglioside-GM.sub.2, Monosialoganglioside-GM.sub.3,
Lipids, Transferrin, B-Cyclodextrin, Ascorbate, Fetuin, Heparin,
2-Mercaptoethanol, Horse Serum, DMSO, Chicken Serum, Goat Serum,
Rabbit Serum, Human Serum, MIP-18, MIP-2, MCP-1, MCP-5, NGF, NGF-B,
Pituitary Extract, Stromal Cell Factor, Conditioned Medium,
Hybridoma Medium, d-Aldosterone, Dexamethasone, DHT, B-Estradiol,
Glucagon, Insulin, Progesterone, Prostaglandin-D.sub.2,
Prostaglandin-E1, Prostaglandin-E.sub.2, Prostaglandin-F.sub.2,
Serum-Free Medium, Gene Therapy Medium, MDBK-GM Medium, QBSF-S1,
Endothelial Medium, Keratinocyte Medium, Melanocyte Medium,
Interleukin-10, Epidermal Growth Factor, Heparin Binding Epidermal
Growth Factor, bFGF, Gly-His-Lys, Insulin Like Growth Factor-II,
IGBFBP/IGF-I Complex, C10, Cytokine Induced Neutrophil
Chemoattractant 2, Cytokine Induced Neutrophil Chemoattractant 2B,
Cytokine Induced Neutrophil Chemoattractant 1, Cytokine Responsive
Gene-2, Endothelial Cell Growth Supplement, Interferon A,
Interferon A/D, Interferon B, Interferon Inducible Protein-10,
Leptin, Methylcellulose, Keratin Sulphate Proteoglycam, Laminin and
Chondroitin Sulphate A.
58. The method of claim 50, wherein said individual members of said
library of transfected cells are separated into separate wells of
one or more multi-well plates.
59. The method of claim 50, wherein said promoters are specifically
expressed in primary or progenitor cells selected from the group
consisting of endodermal cells, mesodermal cells, ectodermal cells,
brain cells, heart cells, liver cells, skin cells, pancreatic
cells, blood cells, reproductive cells, nerve cells, sensory cells,
vascular cells, skeletal cells, immune cells, lung cells, muscle
cells, and kidney cells.
60. The method of claim 50, wherein said totipotent, nearly
totipotent, or pluripotent stem cells, or cells therefrom, are
primate embryonic stem cells.
61. The method of claim 60, wherein said primate embryonic stem
cells are Cyno-1 cells.
62. The method of claim 50, wherein said totipotent, nearly
totipotent, or pluripotent stem cells, or cells therefrom, are
murine embryonic stem cells.
63. The method of claim 62, wherein said murine embryonic stem
cells each contain a gene-trap vector insertion.
64. The method of claim 63, wherein said murine embryonic stem
cells comprise members of OmniBank.RTM. and/or ES cell mutants
corresponding to a gene-trap sequence tag (GTST) reference
library.
65. The method of claim 50, further comprising: isolating primary
and/or progenitor cells from reference tissues and placing said
primary and/or progenitor cells into separate vessels of a
microarray thereby forming a control reference library; and after
exposing said separate vessels of totipotent, nearly totipotent, or
pluripotent stem cells, or cells therefrom, to the said one or more
putative differentiation-inducing compounds either simultaneously
or sequentially; comparing said individual totipotent, nearly
totipotent, or pluripotent stem cells, or cells therefrom, or
groups of cells, to said reference library in order to evaluate the
differentiation of said individual cells or groups of cells.
66. The method of claim 65, wherein said reference library of
primary cells is constructed in one or more multi-well plates.
67. The method of claim 65, wherein said primary and/or progenitor
cells for said reference library include one or more of brain
cells, heart cells, liver cells, skin cells, pancreatic cells,
blood cells, reproductive cells, nerve cells, sensory cells,
vascular cells, skeletal cells, immune cells, lung cells, muscle
cells, and kidney cells.
68. The method of claim 65, wherein said totipotent, nearly
totipotent, or pluripotent stem cells, or cells therefrom, and said
reference library cells are compared using functional assays
specific for the particular primary cells in the reference
library.
69. The method of claim 68, wherein said functional assays measure
the production of enzymes or metabolites produced by said reference
primary and/or progenitor cells.
70. The method of claim 65, wherein said exposed totipotent, nearly
totipotent, or pluripotent stem cells, or cells therefrom, and said
reference library cells are compared by testing for the presence of
specific cell surface markers or receptors.
71. The method of claim 70, wherein the presence of said cell
surface markers is detected using labeled antibodies or ligands
specific for said cell surface markers or receptors.
72. The method of claim 1, wherein said screening step comprises
making multiple measurements over a course of time to identify
agents or conditions that promote increased survival of the cells
over the period of the assay.
73. The method of claim 1, wherein said screening step comprises
making multiple measurements over a course of time to identify
agents or conditions that promote an increase in cell number over
the period of the assay.
74. A library of two or more gene trap stem cell lines used
simultaneously together to screen to detect agents or conditions
that affect differentiation, survival, or proliferation of the stem
cells.
75. The library of claim 74, wherein one or more of said gene trap
stem cell lines is uni-nodal; i.e., contains a reporter gene that
is activated in only one cell type upon differentiation or partial
differentiation of the stem cells.
75. The library of claim 74, wherein one or more of said gene trap
stem cell lines is multi-nodal; i.e., contains a reporter gene is
activated in two or more different cell types upon differentiation
or partial differentiation of the stem cells.
76. A method for inducing differentiation of a stem cell to form
cells of mesodermal lineage, comprising exposing the stem cells to
Flt-3.
77. The method of claim 76 wherein said cells of mesodermal lineage
are vascular endothelial cells.
78. A method for inducing differentiation of a stem cell to form
cells of mesodermal and neural lineage, comprising exposing the
stem cells to TGF-beta-1.
79. A method for inducing differentiation of a stem cell to form
cells selected from the group consisting of cells of endothelial
lineage, and cells of endodermal lineage or appearance, comprising
exposing the stem cells to tenascin.
80. A method for inducing differentiation of a stem cell comprising
exposing the stem cells to Tie-1.
81. The method of claim 80, further comprising exposing the stem
cells to a Fc moiety.
82. A method for inducing differentiation of a stem cell to form
fibroblasts and/or other cells of connective tissue comprising
exposing the stem cells to BMP-2.
83. A method for inducing differentiation of a stem cell to form
myocardial cells comprising exposing the stem cells to endothelial
inducer cells.
84. A method for inducing differentiation of a stem cell to form
cells of mesodermal lineage comprising exposing the stem cells to
fibroblast inducer cells.
84. The method of claim 84, further comprising exposing the stem
cells to FGF-4 and TGF-beta-1.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to methods for the in vitro
culture and differentiation of totipotent, nearly totipotent, and
pluripotent cells, and cells derived therefrom. Examples of such
cells are embryonic cells, embryonic stem cells, embryonic germ
cells, embryoid bodies, inner cell mass cells, morula-derived
cells-derived cells, non-embryonic stem cells of embryonic, fetal,
and adult animals, such as mesenchymal, hematopoietic, and neuronal
stem cells, and cells derived from any of these.
[0002] In one aspect, the invention provides efficient,
high-throughput assays for screening and identifying chemical and
biological agents and physical conditions that may be used to
induce and direct the differentiation of totipotent, nearly
totipotent, and pluripotent cells, and cells therefrom along
particular developmental lineages. Examples of such
differentiation-inducing agents and conditions are growth factors,
cytokines and extracellular matrix components, cell-cell
interactions, environmental conditions (temperature, oxygen
pressure, etc.), and other extracellular factors or components, and
combinations thereof, to which the target stem cells may be exposed
simultaneously or sequentially to induce and direct
differentiation.
[0003] In another aspect, the invention provides a means of making
genetically modified stem cell lines, e.g., gene trap stem cell
lines, that facilitate the production, isolation, and therapeutic
use of differentiated cell types for cell therapy.
[0004] In another aspect, the invention provides a means of
producing and isolating particular types of cells for animal
testing and cell therapy.
[0005] In another aspect, the invention encompasses compositions of
growth factors, cytokines, and/or other chemical and biological
differentiation-inducing agents, alone or in combination, that are
identified by the methods described herein, and their use to direct
the development of characterized cell populations and tissues from
totipotent, nearly totipotent, and pluripotent cells, and cells
therefrom, for use in treatments, transplantation therapies, and
drug discovery, including the discovery of novel cancer targets and
therapies.
BACKGROUND OF THE INVENTION
[0006] The past decade has been characterized by significant
advances in the science of cloning, and has witnessed the birth of
a cloned sheep, i.e. "Dolly" (Roslin Bio-Med), a trio of cloned
goats named "Mira" (Genzyme Transgenics) and over a dozen cloned
cattle (Advanced Cell Technology or ACT). Most recent additions to
the clone family include pigs (PPL Therapeutics) and mice
(University of Hawaii Medical School). Scientists at ACT have also
demonstrated successful cross-species nuclear reprogramming by the
birth of a cloned guar produced using a bovine recipient oocyte.
For example, see U.S. patent application Ser. No. 09/685,062,
incorporated by reference herein in its entirety. Furthermore,
cloning technology has also advanced such that a mammal may now be
cloned using the nucleus from an adult, differentiated cell, which
scientists now know undergoes "reprogramming" when it is introduced
into an enucleated oocyte. See U.S. Pat. No. 5,945,577,
incorporated herein by reference in its entirety.
[0007] The showing that an embryo and embryonic stem cells may be
generated using the nucleus from an adult differentiated cell has
exciting implications for the fields of organ, cell and tissue
transplantation. There are currently thousands of patients waiting
for a suitable organ donor, who face the problems of both
availability and incompatibility in their wait for a transplant. By
using a differentiated cell from a patient in need of a transplant
to generate embryonic stem cells, and inducing these to
differentiate into characterized populations of the cell type
required in the transplant, the problem of transplantation
rejection and the dangers of immunosuppressive drugs could be
precluded. This prospect is now known to many as "therapeutic
cloning," or "adult cell reprogramming" so as to distinguish it
from "reproductive cloning" and provides a moral boundary as the
reach of cloning extends toward the realm of human beings. Lanza et
al., September 1999, Human therapeutic cloning, Nat. Med. 5(9):
975-7.
[0008] Conscious of the promise of therapeutic cloning, scientists
are seeking to understand how to efficiently direct the
differentiation of totipotent and pluripotent stem cells into
particular cell types and tissues, while at the same time deterring
their differentiation into unwanted cells and tissues. Controlled,
specific direction of cell differentiation will come from
deciphering the factors and signals that control embryonic
development. The alternative, e.g., the random differentiation of
embryonic cells and subsequent dissection of desired tissues, is
both impractical and morally unacceptable for human therapy.
[0009] As used herein, a "stem cell" is a cell that has the ability
to divide for indefinite periods in culture and to give rise to
daughter cells of one or more specialized cell types.
[0010] As used herein, an "embryonic stem cell" (ES-cell) is a cell
line with the characteristics of the murine embryonic stem cells
isolated from morulae or blastocyst inner cell masses (as reported
by Martin, G., Proc. Natl. Acad. Sci. USA (1981) 78:7634-7638; and
Evans, M. and Kaufman, M., Nature (1981) 292: 154-156) i.e., ES
cells are immortal and capable of differentiating into all of the
specialized cell types of an organism, including the three
embryonic germ layers, all somatic cell lineages, and the germ
line.
[0011] As used herein, an "embryonic stem-like cell" (ES-like cell)
is a cell of a cell line isolated from an animal inner cell mass or
epiblast that has a flattened morphology, prominent nucleoli, is
immortal, and is capable of differentiating into all somatic cell
lineages, but when transferred into another blastocyst typically
does not contribute to the germ line. An example in the primate "ES
cell" reported by Thomson et al. (Proc. Natl. Acad. Sci. USA.
(1995) 92:7844-7848)
[0012] As used herein, "inner cell mass-derived cells" (ICM-derived
cells) are cells derived from isolated ICMs or morulae before they
are passaged to establish a continuous ES or ES-like cell line.
[0013] As used herein, an "embryonic germ cells" (EG cells) is a
cell of a line of cells obtained by culturing primordial germ cells
in conditions that cause them to proliferate and attain a state of
differentiation similar, though not identical to embryonic stem
cells. Examples are the murine EG cells reported by Matsui, et al,
1992, Cell 70: 841-847 and Resnick et al, Nature. 359: 550-551. EG
cells can differentiate into embryoid bodies in vitro and form
teratocarcinomas in vivo (Labosky et al., Development (1994)
120:3197-3204). Immunohistochemical analysis demonstrates that
embryoids produced by EG cells contain differentiated cells that
are derivatives of all three embryonic germ layers (Shamblott et
al., Proc. Nat. Acad. Sci. U.S.A. (1998) 95:13726-13731).
[0014] As used herein, a "totipotent" cell is a stem cell with the
"total power" to differentiate into any cell type in the body,
including the germ line following exposure to stimuli like that
normally occurring in development. An example of such a cell is an
ES cell, an EG cell, an ICM-derived cell, or a cultured cell from
the epiblast of a late-stage blastocyst.
[0015] As used herein, a "nearly totipotent cell" is a stem cell
with the power to differentiate into most or nearly all cell types
in the body following exposure to stimuli like that normally
occurring in development. An example of such a cell is an ES-like
cell. %
[0016] As used herein, a "pluripotent cell" is a stem cell that is
capable of differentiating into multiple somatic cell types, but
not into most or all cell types. This would include by way of
example, but not limited to, mesenchymal stem cells that can
differentiate into bone, cartilage and muscle; hemotopoietic stem
cells that can differentiate into blood, endothelium, and
myocardium; neuronal stem cells that can differentiate into neurons
and glia; and so on.
[0017] As used herein, "differentiation" refers to a progressive,
transforming process whereby a cell acquires the biochemical and
morphological properties necessary to perform its specialized
functions.
[0018] As used herein, a "marker" is a characteristic or feature of
a cell that is indicative of a particular cellular state.
Typically, a marker is a biochemical entity that changes state in a
detectable manner when the cell enters or leaves a particular
state. For example, a marker may be a DNA sequence encoding a
product that is detectable (e.g., a specific mRNA, or a fluorescent
or antigenic protein) or has detectable activity (e,g., a protein
conferring antibiotic resistance or a chromogenic enzyme such as
lacZ). When copies of the marker DNA sequence are randomly inserted
into the genomic DNA of a cell, some copies may be inserted
proximal to a promoter in the correct orientation and in-frame such
that activation of the promoter results in transcription of the
marker DNA sequence and synthesis of the detectable product that it
encodes. Detection of the marker then identifies the cell as one
that contains the marker gene in a transcriptionally active genetic
locus. The term "marker" as used herein may refer to a marker gene,
or to a marker RNA or protein encoded by such a gene.
[0019] Directed Differentiation of Stem Cells
[0020] Totipotent and nearly totipotent embryo-derived stem cells
can be induced to differentiate into a wide variety of cell types,
some of which are needed for cell therapy. For example, Anderson et
al. demonstrated that inner cell masses (ICM) and embryonic discs
from bovine and porcine blastocysts will develop into teratomas
containing differentiated cell types from ectodermal, mesodermal
and endodermal origins when transplanted under the kidney capsule
of athymic mice. Animal Repro. Sci. 45: 231-240 (1996). Thomson et
al. reported that primate ES cells are capable of differentiating
into trophoblast and derivatives of the three embryonic germ
layers, and describe transplanting primate ES cells into muscles of
immunodeficient mice to generate teratomas that also contain cells
of the three embryonic germ layers, including tissues resembling
neural tube, embryonic ganglia, neurons, and astrocytes (APMIS
(1998) 106(1):149-156). ES cells of mice (Lee et al., Nature
Biotech. (2000) 18:675-679), cynomolgus monkeys (Macaca
fascicularis) (Cibelli et al., Science (2002) 295:819), and humans
(Zhang et al., Nature Biotech. (2001) 19:1129-1133) can be cultured
in vitro to generate embryoids that contain cells of all three germ
layers, including neural precursor cells that test positive for
nestin (an intermediate filament protein produced in the developing
central nervous system and widely used as a marker for
proliferating neural progenitor cells in the nervous system).
Pluripotent stem cells can be isolated from ES and EG cell-derived
teratomas and embryoids and exposed to conditions that induce them
to differentiate into specific cell types that are useful for cell
therapy. For example, nestin-positive neural stem cells isolated
from human embryoids can be cultured under conditions that induce
their differentiation into the three major cell types of the
central nervous system (see Zhang et al. (2001) p.1130).
[0021] The foregoing reports describe the derivation of precursor
or differentiated cells that appear to arise randomly or
spontaneously in embryoids and teratomas generated from totipotent
ES and EG cells. Production of a characterized population of
differentiated cells by these methods therefore requires isolating
the differentiated cells of interest, or their precursors, from
other types of cells in an embryoid or teratoma. Presently, there
is strong interest in identifying chemical, biological, and
physical agents or conditions that induce totipotent or nearly
totipotent cells such as ES and EG cells to differentiate directly
into the desired differentiated cells, in order to develop
efficient methods for producing characterized populations of
differentiated cells that are useful for cell therapy.
[0022] In U.S. Pat. No. 5,733,727, Field described plating murine
ES cells onto uncoated petri dishes and culturing them in medium
that is free of leukemia inhibitory factor (LIF), an inhibitor of
differentiation, to generate patches of cardiomyocytes that exhibit
spontaneous contractile activity (col. 12, lines 63-67). Field also
described a useful method for purifying cells induced to
differentiate into a specific cell type from other types of cells
present in the culture: the parental ES cells are cotransfected
with a pGK-HYG (hygromycin) plasmid and a plasmid containing a
MHC-neo.sup.r fusion gene--an .alpha.-cardiac myosin heavy chain
(MHC) promoter operably linked to a neo.sup.r gene that confers
resistance to neomycin. The PGK-HYG plasmid provides selection for
transfected cells, while the MHC-neo.sup.r gene permits a second
round of selection of the differentiated cells--incubation in the
presence of G418 eliminates non-cardiomyocyte cells in which the
MHC promoter is inactive (see col. 12, lines 63-67). The disclosure
of U.S. Pat. No. 5,733,727 is incorporated herein by reference in
its entirety.
[0023] Schuldiner et al. described a systematic approach to
analyzing the differentiation of ES-derived cells in response to
different growth factors. They cultured human ES cells to generate
embryoids, dissociated the embryoids and cultured the cells as a
monolayer in the presence of one of eight different growth factors.
The differentiation induced by the growth factors was examined by
monitoring changes in the cells' morphologies, and by RT-PCR
(reverse transcription--polymerase chain reaction) analysis of the
expression of a panel of 24 cell-specific genes in the parental ES
cells, embryoid cells, and the dissociated embryoid cells cultured
in the presence or the absence of one of the eight growth factors.
Schuldiner et al. reported that each of the growth factors appeared
to induce expression of different subset of the 24 marker genes
that were analyzed; and that the growth factor-treated cultures
were relatively homogenous, often containing only one or two cell
types, whereas the dissociated embryoid cells cultured in the
absence of a growth factor spontaneously differentiated into many
different types of colonies. The growth factors appeared to act
more by inhibiting than by inducing the differentiation of specific
cell types, and none of the growth factors tested directed a
completely uniform and singular differentiation of cells, and
suggesting that direction of formation of specific cell types will
require combinations of factors including those that inhibit
undesired pathways and those that induce differentiation of
specific cell types. (See Proc. Natl. Acad. Sci. USA (2000) 97(21):
11307-12). Paquin et al. described culturing murine P19 ES cells
under conditions resulting in formation of aggregates of cells,
some of which differentiated into beating cardiomyocytes (Proc.
Nat. Acad. Sci. (2002) 99(14):9550-9555). Reubinoffet al. described
manipulating the conditions in which human ES cells were cultured
to induce their differentiation directly into neural precursors
that could then be induced to differentiate into derivatives of the
three neural lineages, neuronal cells, glial cells, and astrocytes
(Nature Biotechnology (2001) 19:1134-1139). Kelly et al. have shown
that changes in gene expression in ES cells in response to retinoic
acid are highly reproducible (Mol. Reprod. Dev. (2000)
56(2):113-23), a result that implies that growth factor-directed
differentiation of embryonic cells is dependably reproducible.
[0024] Other groups have had success in using a negative approach
to identify factors necessary for the differentiation of ES cells
into certain cell types. For instance, Henkel and colleagues
reported that the transcription factor PU.1 is essential for
macrophage development from embryonic stem cells by showing that ES
cells containing a homozygous knockout of the PU.1 gene failed to
differentiate into macrophages (see Henkel et al., Blood (1996)
88(8): 2917-26). Similarly, Dunn and colleagues demonstrated that
knockout embryoid bodies containing a targeted disruption of the
phosphatidylinositol glycan class A (Pig-a) gene failed to develop
secondary hematopoietic colonies and demonstrated a grossly
aberrant morphology (see Dunn et al., Proc. Natl. Acad. Sci. USA
(1996) 93(15): 7938-43).
[0025] Directed differentiation has also been demonstrated
successfully in pluripotent adult stem cells. For instance, U.S.
Pat. No. 5,942,225 to Bruder et al. describes the lineage-directed
induction of human mesenchymal stem cell differentiation by
exposing such stem cells to a bioactive factor or combination of
factors effective to induce differentiation either ex vivo or in
vivo. Mesenchymal stem cells are more differentiated than embryonic
stem cells and only differentiate into lineages including
osteogenic, chondrogenic, tendonogenic, ligamentogenic, myogenic,
marrow stromagenic, adipogenic and dermogenic lineages. Similarly,
U.S. Pat. No. 5,851,832 to Weiss et al. describes the in vitro
proliferation and differentiation of neural stem cells following
exposure of the cells to various growth factors. Such stem cells
are limited in their differentiation potential, producing only
neurons and glial cells, including astrocytes and oligodendrocytes
(see also Brannen et al., Neuroreport (2000) 11(5): 1123-8; Lillien
et al., Dev. (2000) 127: 4993-5005).
[0026] The studies described above have shown that totipotent,
nearly totipotent, and pluripotent stem cells can be induced to
differentiate into specific cell types by manipulating the
concentration of growth factors and cytokines in the medium in
which they are cultured. Other examples of growth factor-induced
differentiation include induction of stem cells to become
macrophages, mast cells or neutrophils by IL-3 (Wiles et al.,
Development (1991) 111:259-267); the direction of cells to the
erythroid lineage by IL-6 (Biesecker et al., Exp. Hematol. (1993)
21: 774-778); induction of neuronal differentiation by retinoic
acid (Slager et al., Dev. Genet. (1993) 14: 212-224; Bain et al.,
Dev. Biol. (195) 168:342-357); and induction of myogenesis by
transforming growth factor (Rohwedel et al., Dev. Biol.
(1994)164,87-101). In the latter examples, the inducing agents were
not directly applied to ES cells or cells directly derived from the
embryo, but rather to aggregates of ES cells or to embryoids.
[0027] In addition to manipulating the concentration of growth
factors and cytokines, totipotent and pluripotent stem cells may be
induced to differentiate into specific cell types by co-culturing
them with cells of a different type. For example, Kaufman et al.
(U.S. Pat. No. 6,280,718) showed that human ES cells differentiate
into hematopoietic precursor cells when cultured on a feeder cell
layer of mammalian stromal cells (see col. 5, line 7, to col. 6,
line 26). The disclosure of U.S. Pat. No. 6,280,718 is incorporated
herein by reference in its entirety. Similarly, Kawasaki et al.
have induced the differentiation of cynomolgus monkey ES cells into
dopaminergic neurons and pigmented epithelial cells by culturing
them on a feeder layer of murine stromal cells (see Proc. Natl.
Acad. Sci. USA (2002) 99(3):1580-85).
[0028] As shown by the reports described above, research groups'
attempts to identify the agents or conditions that induce the
differentiation of totipotent and pluripotent stem cells into
specific cell types generally involve exposing the stem cells to
one or two solutions containing a relatively small number of growth
factors or cytokines, and monitoring to see if the stem cells
differentiate to acquire a morphology and/or to express a marker
gene that is characteristic of a specific cell type.
[0029] At present, there is a need for a systematic, large-scale,
screening assay to efficiently identify the combinations of
biological, biochemical, and physical agents or conditions that
act, simultaneously or sequentially, to induce the differentiation
of totipotent, nearly totipotent, or pluripotent stem cells into a
large number of different, specific cell types.
[0030] Also needed are means for efficiently identifying, analyzing
and characterizing marker genes and gene products that specifically
mark key regulatory steps associated with the induction of
differentiation of such stem cells into each of the important
specific cell types.
[0031] There is also a need for an efficient means for producing
and purifying characterized populations of differentiated cells
that are suitable and useful for cell therapy, and for testing
these in animal models.
[0032] The present invention accomplishes these ends, without being
limited thereto.
[0033] Differentiation Pathways in Oncogenesis
[0034] Many molecular events in oncogenesis are a recapitulation or
mutation of events that normally occur in differentiation. In this
respect, in many cases oncogenesis reflects a reversal of terminal
differentiation utilizing, at least in part, pathways used in
normal development. Control of cell growth and differentiation by
extracellular signals often involves growth factor binding to high
affinity transmembrane receptors such as the receptor tyrosine
kinases (RTKs) For example, Recently Sakamoto et al, 2001, (Oncol.
Rep. 8: 973-80) reported that nerve growth factor and its
low-affinity receptor p75NGFR play a role in breast cancer, Gmyrek
et al, 2001 (Am. J. Pathol. 159: 579-90) described the role of
hepatocyte growth factor/scatter factor ((HGF/SF) that binds the
Met receptor and promotes the differentiation of epithelial cells
in prostate, kidney, and hepatocellular carcinoma, similarly,
mutations in the Ret receptor has been implicated in multiple
endocrine neoplasias, the kit receptor in mastocytomas and
gastrointestinal tumors, the Flt-3 ligand that plays a role in
hematopoietic differentiation has been implicated in neural
crest-derived tumors (Timeus et al, 2001, Lab. Invest. 81:
1025-1037), FGF-1 and -2 in pancreatic malignancy (El-Hariry et al,
2001, Br. J. Cancer, 84: 1656-63), HB-EGF in colon cancer (Ito et
al, 2001, Anticancer Res. 21: 1391-4), Oncostatin M in breast
cancer, Glypicans in breast cancer (Matsuda et al, 2001, Cancer
Res. 61: 5562-9), and Yiu et al, 2001 (Am. J. Pathol. 159: 609-22)
described the role of the extracellular matrix component SPARC in
the apoptosis pathway in ovarian cancer. These only a few examples
of the many extracellular components that are important in the
differentiation of a particular cell type, and also play a role in
cancer. Surprisingly, few assays for antitumor agents, or assays
for novel targets in cancer therapy have been based on the
identification of factors influencing early differentiation
pathways. The present invention also provides means for efficiently
screening many combinations of biological, biochemical, and
physical agents or conditions to identify treatments that may
induce cancerous cells to undergo differentiation and inhibit their
proliferation.
BRIEF DESCRIPTION OF THE FIGURES
[0035] FIG. 1A is a photograph that shows primate Cyno-1FF ES-like
cells conditioned to grow on tissue culture dishes without feeder
fibroblasts (1.times.).
[0036] FIG. 1B shows Cyno-1 FF cells at a higher magnification,
showing the typical morphology of ES-like cells (40.times.).
[0037] FIG. 2: Table 1 identifies the factors added to each of the
wells of the duplicate 24-well plates of Example 2.
[0038] FIG. 3 is a photograph showing Cyno-1 FF cells that were
exposed to Flt-3 ligand.
[0039] FIG. 4 shows mesoderm and cells with the morphology of
nestin positive neuronal stem cells obtained by culturing Cyno-1 FF
cells in the presence of TGF beta-1.
[0040] FIG. 5 shows cells having the appearance of endodermal
precursor cells obtained by culturing Cyno-1 FF cells in the
presence of the extracellular matrix protein tenascin.
[0041] FIG. 6 shows Cyno-1 FF cells exposed to a chimeric protein
made from the receptor for Tie-1 and an immunoglobulin Fc
region.
[0042] FIG. 7 shows fibroblast-like connective tissue cells
produced by culturing Cyno-1 FF cells in the presence of BMP-2.
[0043] FIG. 8: Table 2 identifies the primers that were used to
detect expression of cell type-associated genes by RT-PCR, and the
expected sizes of the DNA fragments produced by the RT-PCR
reactions.
[0044] FIG. 9 shows examples of the results of RT-PCR analysis of
cells from four different wells, each containing a different
inducing agent (see Example 2). The figure shows photographs of the
lanes of electrophoretic gels in which the DNA molecules produced
by RT-PCR were separated, stained with ethidium bromide, and
illuminated with uv light.
[0045] FIG. 10 shows the detection of desmin by ICC in Cyno-1 FF
cells exposed to a differentiation-inducing agent (see Example
3).
[0046] FIG. 11 shows the detection of nestin by ICC in Cyno-1 FF
cells exposed to a differentiation-inducing agent (see Example
3).
[0047] FIGS. 12A and 12B are phase contrast photographs of the
cells in well #16 of Example 5 that were exposed to IL-1-alpha.
[0048] FIG. 12A (on left): The arrowhead points to a beating
myocardial cell.
[0049] FIG. 12B (on right): The arrowhead points to an endothelial
cell adjacent to myocardial cells.
[0050] FIG. 13: Table 3 identifies the combinations of putative
differentiation-inducing agents added to the wells of the 24 well
plates in which murine ES cells were cultured as described in
Example 6.
[0051] FIG. 14 shows the detection of desmin by ICC in murine ES
cells cultured in TGF-beta-1 and FGF-4 for five days on type I
collagen and human plasma fibronectin (see Example 6).
[0052] FIG. 15 shows the detection of X-gal staining of cells of
the murine gene trap ES cell line K18E2 that were cultured for five
days on type I collagen and human plasma fibronectin in the
presence of TGF-beta-1 and FGF-4 (see Example 7). Detection of
expression of the marker beta-galactosidase gene in the gene trap
ES cells indicates that the cells were induced to
differentiate.
[0053] FIG. 16 shows the detection of beta-galactosidase by ICC
(using antibody to beta-galactosidase) in cells of murine gene trap
ES cell line M7H7 that were cultured for five days on type I
collagen and human plasma fibronectin in the presence of TGF-beta-1
and FGF-4. Nuclei are co-visualized by DAPI staining.
[0054] FIG. 17 shows the detection of beta-galactosidase by ICC in
cells of murine gene trap ES cell line K18E2 that were cultured for
five days on type I collagen and human plasma fibronectin in the
presence of FGF-4.
[0055] FIG. 18 shows the presence of .beta.-galactosidase in K18E2
cells that were cultured with FGF-4 and TGF-.beta.1 on inducer
fibroblasts for 5 days, then sub-cultured for an additional 5 days
with FGF-4 and TGF-.beta.1 alone.
[0056] FIG. 19 shows the presence of .beta.-galactosidase in M7H7
cells that were cultured with FGF-4 and TGF-.beta.1 on inducer
fibroblasts for 5 days, then sub-cultured for an additional 5 days
with FGF-4 and TGF-.beta.1 alone.
[0057] FIG. 20: shows the presence of .beta.-galactosidase in K18E2
cells that were cultured with FGF-4 and TGF-.beta.1 in the absence
of inducer fibroblasts, and then sub-cultured for 5 more days in
the same conditions.
[0058] FIG. 21 shows the presence of .beta.-galactosidase in M7H7
cells that were cultured with FGF-4 and TGF-.beta.1 in the absence
of inducer fibroblasts, and then sub-cultured for 5 more days in
same conditions.
DESCRIPTION OF THE INVENTION
[0059] An object of the present invention is to provide a
high-throughput screening assay for efficiently identifying
chemical, physical, and biological agents and/or conditions, and
combinations of such agents and/or conditions, that induce or
direct the differentiation of totipotent, nearly totipotent, or
pluripotent stem cells, and cells therefrom into a large number of
different, specific cell types, including cell types that are
useful for cell therapy.
[0060] Another object of the present invention is to provide
efficient means for identifying and characterizing biochemical
markers in cells that are associated with the series of regulatory
steps or "nodes" in the branching pathways by which totipotent,
nearly totipotent, or pluripotent stem cells, and cells therefrom
differentate into a large number of different, specific cell types,
including cell types that are useful for cell therapy.
[0061] Another object of the present invention is to provide
efficient means for producing totipotent, or pluripotent stem
cells, and cells therefrom that are genetically modified to
facilitate the production, isolation, and therapeutic use of
differentiated cell types for cell therapy.
[0062] In one aspect, the invention includes assays for identifying
chemical and biological agents and physical conditions which may be
used to direct the differentiation of totipotent, nearly
totipotent, and pluripotent cells, and cells therefrom along a
particular developmental lineage. Examples of such
differentiation-inducing chemical and biological agents and
physical conditions are growth factors, cytokines and extracellular
matrix components, cell-cell interactions, environmental conditions
(temperature, oxygen pressure, etc.), and other extracellular
factors or components, and combinations thereof, to which the
target cells may be exposed simultaneously or sequentially.
Examples of biological agents that can be used as putative
differentiation-inducin- g agents include living or dead cells of
all types, as well as portions or fractions of any cells, including
compositions comprising organelles, internal and external cell
membranes, membrane-associated proteins, soluble proteins, protein
complexes, complexes of proteins and other molecular classes,
including lipids, carbohydrates, and nucleic acids, etc. Methods
for fractionating cells to prepare fractions that may be used as
biological agents that are putative differentiation-inducing agents
are well known. Other biological agents useful as
differentiation-inducing agents are cell culture-conditioned
medium, and extracts or fractions of natural or artificial
tissues.
[0063] In another aspect, the invention provides means of making
gene trap stem cell lines that have DNA encoding a detectable
marker inserted as a marker gene in a genetic locus that is
activated when the cells differentiate. The DNA encoding the gene
trap marker may be inserted in-frame with correct orientation at a
site such that it is expressed and the marker is produced when the
genetic locus in which it is inserted is activated. The inserted
coding sequence then operates as a marker permitting detection of
the differentiation of the stem cells. DNA encoding
beta-galactosidase is an example of a commonly used gene trap
marker suitable for the invention.
[0064] Another aspect of the present invention to provide efficient
means for producing totipotent, or pluripotent stem cells, and
cells therefrom that are genetically modified to facilitate the
production, isolation, and therapeutic use of differentiated cell
types for cell therapy.
[0065] In another aspect, the invention provides a means of
isolating particular types of cells for animal testing and cell
therapy.
[0066] In another aspect, the invention encompasses compositions of
growth factors, cytokines, and/or other differentiation-inducing
agents, alone or in combination, that are identified by the methods
described herein, and their use to direct the development of
characterized cell populations and tissues from totipotent, nearly
totipotent, and pluripotent cells, and cells therefrom, for use in
treatments, transplantation therapies, and drug discovery,
including the discovery of novel cancer targets and therapies.
[0067] Nuclear transfer is a useful method for generating
totipotent, nearly totipotent, or pluripotent stem cells that can
be used in the methods of the invention for screening agents and
conditions that induce and direct stem cells differentiation. The
nuclear transfer methods useful for generating stem cells for the
screening methods of the present invention are the same as those
for generating totipotent, nearly totipotent, or pluripotent stem
cells that differentiate into cells that are useful for cell
therapy. Such methods are described in the co-pending International
Application filed on Jul. 18, 2002, based on U.S. Provisional
Application No. 60/305,904 and assigned to Advanced Cell
Technology, the contents of which are incorporated herein in their
entirety, nuclear transfer can also be used to generate
[0068] Stem Cells:
[0069] The assays of the invention may be performed with any
appropriate totipotent, nearly totipotent, or pluripotent stem
cells, and cells therefrom. Such cells include inner cell mass
(ICM) cells, embryonic stem (ES) cells, embryonic germ (EG) cells,
embryos consisting of one or more cells, embryoid body (embryoid)
cells, morula-derived cells, as well as multipotent partially
differentiated embryonic stem cells taken from later in the
embryonic development process, and also adult stem cells, including
but not limited to nestin positive neural stem cells, mesenchymal
stem cells, hematopoietic stem cells, pancreatic stem cells, marrow
stromal stem cells, endothelial progenitor cells (EPCs), bone
marrow stem cells, epidermal stem cells, hepatic stem cells and
other lineage committed adult progenitor cells.
[0070] Totipotent, nearly totipotent, or pluripotent stem cells,
and cells therefrom, for use in the present invention can be
obtained from any source of such cells. One means for producing
totipotent, nearly totipotent, or pluripotent stem cells, and cells
therefrom, for use in the present invention is via nuclear transfer
into a suitable recipient cell as described in U.S. Ser. No.
09/655,815, the disclosure of which is incorporated herein by
reference in its entirety. Nuclear transfer using an adult
differentiated cell as a nucleus donor facilitates the recovery of
transfected and genetically modified stem cells as starting
materials for the present invention, since adult cells are often
more readily transfected than embryonic cells.
[0071] The methods of the invention may be performed with
totipotent, nearly totipotent, or pluripotent stem cells, and cells
therefrom, of any animal species, including but not limited to
human and non-human primate cells, ungulate cells, rodent cells,
and lagomorph cells. Primate cells with which the invention may be
performed include but are not limited to cells of humans,
chimpanzees, baboons, cynomolgus monkeys, and any other New or Old
World monkeys. Ungulate cells with which the invention may be
performed include but are not limited to cells of bovines,
porcines, ovines, caprines, equines, buffalo and bison. Rodent
cells with which the invention may be performed include but are not
limited to mouse, rat, guinea pig, hamster and gerbil cells.
Rabbits are an example of a lagomorph species with which the
invention may be performed.
[0072] For example, the methods of the invention may be performed
with murine ES cells lines, or with primate ES or EG cell lines. An
example of a primate stem cell line with which the methods of the
invention may be performed is the totipotent non-human primate stem
cell line Cyno-1, which was isolated from the inner cell mass of
parthenogenetic Cynomologous monkey embryos and is capable of
differentiating into all the cell types of the body. Cibelli et al.
(Science (2002) 295:819).
[0073] Genetic Modification of Stem Cells:
[0074] Some embodiments of the invention use stem cells that have
been genetically modified, or a library of such stem cells. For
example, screening to identify agents or conditions that induce
stem cells to differentiate into a large number of different,
specific cell types can be carried out efficiently in a
high-throughput manner using gene trap stem cell libraries, as
discussed below.
[0075] After employing the screening assays of the invention to
identify agents or conditions that induce stem cells to
differentiate into desired cell types, e.g., cells that are useful
for cell therapy, it is an aspect of the present invention to
genetically modify the stem cells (either the gene trap cells, or
unmodified ES cells of the same type), to facilitate the
production, isolation, and therapeutic use of differentiated cell
types for cell therapy.
[0076] For example, stem cells that give rise to differentiated
cells for cell therapy can be genetically modified by correcting
congenital mutations, or by introducing, altering, or deleting one
or more genomic DNA sequences to provide therapeutic benefit to the
patient receiving the cell transplant (gene therapy).
[0077] Nuclear transfer using an adult differentiated cell as a
nucleus donor facilitates the recovery of transfected and
genetically modified stem cells as starting materials for the
present invention, since adult cells are often more readily
transfected than embryonic cells.
[0078] In some instances, these cells may be genetically modified
to express a selectable marker, or engineered with a genetic
modification that renders the cells lineage defective. For
instance, selectable markers may be utilized to further purify
specific cell types from samples of differentiated cells derived
using the methods reported herein. Such methods would include the
use of positive selection wherein the selectable marker is, for
example, the neomycin or hygromycin resistance gene. This allows
the cells that have not differentiated into the chosen cell type to
be killed by G418 in the case of neomycin resistance.
Alternatively, the specific promoter may drive other selection
systems such as a cell surface antigen that allows, for instance,
the isolation of the chosen cells using flow cytometry.
Alternatively, cells may be modified with a suicide gene operably
expressed from a tissue-specific or lineage specific promoter,
i.e., as a supplement to the compounds and combinations identified
using the methods disclosed herein, in order to facilitate the
recovery of desirable cells and tissues.
[0079] Culturing on Serum-Free Medium:
[0080] Embryonic cells have the propensity to differentiate
randomly and rapidly upon removal of LIF (leukemia inhibitory
factor), and the feeder cells normally used to maintain embryonic
cells may produce growth factors or other compounds that could
complicate results (see Reubinoff et al., Nature Biotech. (2000)
18(4): 399-404). Thus, an embodiment of the screening assays may
include adapting the cells to a serum-free medium or, in the case
of some embryo-derived cells, to growth in the absence of a
fibroblast feeder layer in which they do not necessarily need to
proliferate, but in which they will survive and remain responsive
to the test compounds applied. Different serum-free media are known
in the art and may be tested and used with any given cell line in
the methods disclosed herein. For instance, in evaluating the in
vitro growth and differentiation of multipotent stem cells, U.S.
Pat. No. 5,851,832 (herein incorporated by reference) describes the
use of a serum-free medium composed of DMEM/F-12 (1:1) including
glucose (0.6%), glutamine (2 .mu.M), sodium bicarbonate (3 mM), and
HEPES. A defined hormone and salt mixture was used in place of
serum. Wiles et al. describe a serum-free chemically defined medium
(CDM) for studying ES cell differentiation that fails to support
spontaneous differentiation of ES cells while still permitting the
evaluation of differentiation in response to exogenous factors (see
Wiles et al., Exp. Cell Res. (1999) 247(1): 241-8). According to
this group, in the absence of LIF and a feeder layer, ES cells
typically differentiate rapidly, forming predominantly endoderm,
mesoderm and hematopoietic cells. However, in CDM, the cells still
lose their ES cell phenotype but fail to form mesoderm. Rather, the
cells enter a neuroectoderm commitment up to a limited point that
is thought to be a type of "default" pathway that occurs in the
absence of any exogenous differentiation signals.
[0081] Nichols and colleagues report the maintenance of ES cells in
the absence of a feeder layer with a combination of IL-6 plus
soluble IL-6 receptor. Nichols et al., 1994, Derivation of
germ-line competent embryonic stem cells with a combination of
interleukin-6 and soluble interleukin 6 receptor, Exp. Cell Res.
215(1): 237-9. However, this combination activates the same
signaling processes as does LIF, so this medium may not be suitable
to study the putative differentiation inducing factors. Although,
it has been reported that ES cells do differentiate in the presence
of LIF (see Shen et al., Proc. Natl. Acad. Sci. USA (1992) 89:
8240-44). Furthermore, in vivo, LIF is present at the blastocyst
stage of development (Murray et al., Mol. Cell. Biol. (1990) 10:
4953-56). Thus, the response of ICM cells toward LIF may be
regulated temporally and/or spatially in order to permit
development to proceed.
[0082] Another group has isolated an ES cell line that is feeder
cell-independent and LIF-independent, and yet still contributes to
all embryonic germ layers when placed in the environment of a
developing embryo (Berger et al., Growth Factors (1997) 14(2-3):
145-59). However, the cells were isolated by selection through
passage so the mutations that contribute to this self renewal
ability are not known. Nevertheless, one can isolate a similar line
of ES cells to be used in the present invention as an alternative
to developing a specific maintenance medium.
[0083] Another option is to maintain the embryonic cells on a
feeder layer in the presence of LIF until the time of the assay. In
their evaluation of the affects of eight different growth factors
on ES cells, Schuldiner and colleagues transferred the ES cells to
gelatin coated plates for five days to allow for initial
differentiation as aggregates, then replated the cells as a
monolayer wherein the cells were exposed to the test growth
factors. See Schuldiner et al., 2000, supra. A similar approach is
commonly used to direct mouse ES cells in to specific cell types,
such as nerve cells or muscle cells (Slager et al., 1993, supra;
Bain et al., 1995, supra; and Rohwedel et al., 1994, supra).
However, Schuldiner also reported that the cells spontaneously
differentiated into all different cell types in the absence of any
tested growth factor, wherein the samples that were treated with
specific growth factors were more homogenous than the untreated
control. Thus, it may be for any particular assay that the
combination of compounds tested will achieve the directed
differentiation desired in the absence of specific media
formulations that seek to deter differentiation. Indeed,
researchers are finding that the process of directed
differentiation may involve compounds that inhibit certain
developmental pathways either alone or in combination with
inductive compounds.
[0084] Inducers of Differentiation:
[0085] The methods of the invention may be used to screen a wide
variety of compounds and culture conditions to determine their
effect on the differentiation of stem cells. For instance, the
methods may be performed with one or more putative
differentiation-inducing compounds selected from the group
consisting of growth factors, cytokines, factors involved in
cell-to-cell interactions, adhesion molecules, extracellular matrix
components, media components, environmental conditions, etc. Media
components suitable for use include both identified and
unidentified media components; for example, unidentified present in
medium conditioned by cell culture may be used as an inducer of
differentiation. The present invention includes screening to
identify biological compositions that comprise one or more
unidentified agents that induce differentiation, and using known
fractionation and assay methods to isolate the active agent(s).
[0086] The methods and assays of the present invention may also be
used to analyze the differentiation of cells in response to
materials isolated from early stage fetuses or factors or
homogenates or isolated differentiated cells derived therefrom.
Other cells, including primary cells and tissues or isolated cell
lines, may also be screened for their potential to induce the
differentiation of cells according to the disclosed methods and
assays.
[0087] Examples of growth factors, chemokines, and cytokines that
may be tested in the disclosed assays include but are not limited
to the Fibroblast Growth Factor family of proteins (FGF1-23)
including but not limited to FGF basic (146 aa) and it's variants,
FGF acidic, the TGF beta family of proteins including but not
limited to TGF-beta 1, TGF-beta 2, TGF-beta sRII, Latent TGF-beta,
the Tumor necrosis factor (TNF) superfamily (TNFSF) including but
not limited to TNFSF1-18, including TNF-alpha, TNF-beta, the
insulin-like growth factor family incuding but not limited to
IGF-II and their binding proteins including but not limited to
IGFBP-1, II-1 R rp2, IGFBP-5, IGFBP-6, the matrix
metalloproteinases including but not limited to MMP-1, CF, MMP-2,
CF, MMP-2 (NSA-expressed), CF, MMP-7, MMp-8, MMP-10, MMP-9, TIMP-1,
CF, TIMP-2 and other growth factors and cytokines including but not
limited to PDGF, Flt-3 ligand, Fas Ligand, B7-1(CD80), B7-2(CD86),
DR6, IL-13 R alpha, IL-15 R alpha, GRO beta/CXCL2 (aa 39-107), IL
1-18, II-8/CXCL8, GDNF, G-CSF, GM-CSF, M-GSF, PDGF-BB, PDGF-AA,
PDGF-AB, IL-2 sR alpha, IL-2 sR beta, Soluble TNF RII, IL-6 sR,
Soluble gp130, PD-ECGF, IL-4 sR, beta-ECGF, TGF-alpha, TGF-beta
sRII, TGF-beta 5, LAP (TGF-beta 1), BDNF, LIF sR alpha, LIF,
KGF/FGF-7, Pleiotrophin, ENA-78/CXCL5, SCF, beta-NGF, CNTF,
Midkine, HB-EGF, SLPI, Betacellulin, Amphiregulin, PIGF,
Angiogenin, IP-10/CXCL10, NT-3, NT-4, MIP-1 alpha/CCL3, MIP-1
beta/CCL4, I-309/CCL1, GRO alpha/CXCL1, GRO beta/CXCL2, GRO
gamma/CXCL3, Rantes/CCL5, MCP-1/CCL2, MCP-2/CCL8, MCP-3/CCL7,
IFN-gamma, Erythropoietin, Thrombopoietin, MIF, IGF-I, IGF-II,
VEGF, HGF, Oncostatin M, HRG-alpha (EGF Domain), TGF-beta 2, CNTF R
alpha, Tie-2/Fc Chimera, BMP-4, BMPR-IA, Eotaxin/CCL11, VEGF R1
(Flt-1), PDGF sR alpha, HCC-1/CCL14, CTLA-4, MCP-4/CCL13,
GCP-2/CXCL6, TECK/CCL25, MDC/CCL22, Activin A,
Eotaxin-2/MPIF-2/CCL24, Eotaxin-3/CCL-26 (aa 24-94), TRAIL R1
(DR4), VEGF R3 (Fit-4)/SDF-1 alpha(PBSF)/CXCL12, MSP, BMP-2,
HVEMNEGF R2 (KDR), Ephrin-A3, MIP-3 alpha/CCL20, MIP-3 beta/CCL19,
Fractalkine/CX3CL1 (Chemokine Domain), TARC/CCL17, 6Ckine/CCL21,
p75 Neurotrophin R (NGF R), SMDF, Neurturin, Leptin R/Fc Chimera,
MIG/CXCL9, NAP-2/CXCL7, PARC/CCL18, Cardiotrophin-1 (CT-1), GFR
alpha-2, BMP-5, IL-8/CXCL8 (Endothelial Cell Derived), Tie-1, Viral
CMV UL146, VEGF-D, Angiopoietin-2, Inhibin A, TRANCE/RANK L, CD6/Fc
Chimera, CF, dMIP-1 delta/LKN-1/CCL15 (68 aa), TRAIL R3/Fc Chimera,
Soluble TNF RI, Activin RIA, EphA1, ENA-70, ENA-74,
Eotaxin-3/CCL26, ALCAM, FGFR1 alpha (IIIc), Activin B, FGFT1 beta
(IIIc), LIGHT, FGFR2 beta(IIIb), DNAM-1, Follistatin, GFR alpha-3,
gp 130, I-TAC/CXCL11, IFN-gamma R1, IGFBP-2, IGFBP-3, Inhibin B,
Prolactin CF, RANK, FGFR2 beta (IIIc), FGFR4, TrkB, GITR, MSP R,
GITR Ligand, Lymphotactin/XCL1, FGFR2 alpha (IIIc), Activin AB,
ICAM-3 (CD50), ICAM-1 (CD54), TNF RII, L-Selectin (CD62L,
BLC/BCA-1/CXCL13, HCC-4/CCL16, ICAM-2 (CD102), IGFBP-4,
Osteoprotegerin (OPG), uPAR, Activin RIB, VCAM-1 (CD106), CF,
BMPR-II, IL-18 R, IL-12 R beta 1, Dtk, LBP, SDF-1 alpha
(PBSF)/CXCL12 (synthetic), E-Selectin (CD62E), L-Selectin (CD62L),
P-Selectin (CD62P), ICAM-1 (CD54), VCAM-1 (CD106), CD31 (PECAM-1),
hedgehog family of proteins, Interleukin-10, Epidermal Growth
Factor, Heregulin, HER4, Heparin Binding Epidermal Growth Factor,
bFGF, MIP-18, MIP-2, MCP-1, MCP-5, NGF, NGF-B, leptin, Interferon
A, Interferon A/D, Interferon B, Interferon Inducible Protein-10,
Insulin Like Growth Factor-II, IGBFBP/IGF-1 Complex, C10, Cytokine
Induced Neutrophil Chemoattractant 2, Cytokine Induced Neutrophil
Chemoattractant 2B, Cytokine Induced Neutrophil Chemoattractant 1,
Cytokine Responsive Gene-2, and any fragment thereof and their
neutralizing antibodies. The dosage can be in the range of
well-established effective concentrations; for example, dosage can
be in the range of 0.1 to 5 times the maximum value of the
EC.sub.50, the concentration that provokes a response halfway
between baseline and maximum.
[0088] Factors involved in cell-cell interactions that may be
tested include but are not limited to the ADAM (A Disintegrin and
Metalloproteinase) family of proteins including ADAM 1, 2, 3A, 3B,
4-31 and TS1-9, ADAMTSs (ADAMs with thrombospondin motifs),
Reprolysins, metzincins, zincins, and zinc metalloproteinases and
their neutralizing antibodies.
[0089] Adhesion molecules that may be tested include but are not
limited to Ig superfamily CAM's, integrins, cadherins, including
E-, P-, and N-cadherin, and selectins, and their neutralizing
antibodies.
[0090] Nucleic acids that may be tested include but are not limited
to those that encode or block by antisense, ribozyme activity, or
RNA interference transcription factors that are involved in
regulating gene expression during differentiation, genes for growth
factors, cytokines, and extracellular matrix components, or other
molecular activities that regulate differentiation.
[0091] Extracellular matrix component may also induce and direct
the differentiation of stem cells. Members of the tenascin family
are examples of extracellular matrix components that are useful in
directing cell differentiation. There are currently five members of
the family, tenascin-C (simply called tenascin in the examples
below), and tenascins-R, -X, -Y and -W. Tenascin-R is especially
useful in screens for agents that induce cells of the central
nervous system, while tenascins-X and -Y are useful in screens
relating to muscle cells. Tenascin-C is useful in differentiating a
wide array of cell types, including neuronal and endodermal cells.
Agents that block the action of the tenascins, such as neutralizing
antibodies, and proteolytic subunits of the tenascins are also
useful in directing differentiation. The tenascins or their
subunits may be added to the culture substrate prior to the culture
of the cells of interest, added to the media of the cultured cells,
expressed by cells co-cultured with the cells of interest, or
otherwise introduced into contact with the cells.
[0092] Extracellular matrix components that may be tested include
but are not limited to Tenascins, Keratin Sulphate Proteoglycan,
Laminin, Merosin (laminin a2-chain), Chondroitin Sulphate A, SPARC,
beta amyloid precursor protein, beta amyloid, presenilin 1,2,
apolipoprotein E, thrombospondin-1,2, heparin, Heparan Sulphate,
Heparan sulphate proteoglycan, Matrigel, Aggregan, Biglycan,
Poly-L-Ornithine, the collagen family including but not limited to
Collagen I-IV, Poly-D-Lysine, Ecistatin (viper venom), Flavoridin
(viper venom), Kistrin (viper venom), Vitronectin,
Superfibronectin, Fibronectin Adhesion-Promoting peptide,
Fibronectin Fragment III-C, Fibronectin Fragment-30 KDA,
Fibronectin-Like Polymer, Fibronectin Fragment 45 KDA, Fibronectin
Fragment 70 KDA, Asialoganglioside-GM, Disialoganglioside-GOLA,
Monosialo Ganglioside-GM.sub.1, Monosialoganglioside-GM.sub.2,
Monosialoganglioside-GM.sub.3,, Methylcellulose, Keratin Sulphate
Proteoglycam, Laminin and Chondroitin Sulphate A. Extracellular
matrix components can be applied to the culture wells prior to or
after adding the cells. When coating the well surfaces, the
concentration of these components can be in the range of from 1 to
10 mg/ml, or from 0.2 to 50 mg/ml.
[0093] Media components that may be tested include but are not
limited to glucose concentration, lipids, transferrin,
B-Cyclodextrin, Prostaglandin F.sub.2, Somatostatin, Thyrotropin
Releasing Hormone, L-Thyroxine, 3,3,5-Triiodo-L-Thyronine,
L-Ascorbic Acid, Fetuin, Heparin, 2-Mercaptoethanol, Horse Serum,
DMSO, Chicken Serum, Goat Serum, Rabbit Serum, Human Serum,
Pituitary Extract, Stromal Cell Factor, Conditioned Medium,
Hybridoma Medium, d-Aldosterone, Dexamethasone, DHT, B-Estradiol,
Glucagon, Insulin, Progesterone, Prostaglandin-D.sub.2,
Prostaglandin-E.sub.1, Prostaglandin-E.sub.2,
Prostaglandin-F.sub.2, Serum-Free Medium, Endothelial Cell Growth
Supplement, Gene Therapy Medium, MDBK-GM Medium, QBSF-S1,
Endothelial Medium, Keratinocyte Medium, Melanocyte Medium,
Gly-His-Lys, soluble factors that inhibit or interfere with
intracellular enzymes or other molecules including but not limited
to compounds that alter chromatin modifying enzymes such as histone
deacetylases such as butyrate or trichostatin A, compounds that
modulates cAMP, protein kinanse inhibitors, compounds that alter
intracellular calcium concentration, compounds that modulate
phosphatidylinositol.
[0094] Environmental conditions that may be tested include but are
not limited to oxygen tension, carbon dioxide tension, nitric oxide
tension, temperature, pH, mechanical stress, altered culture
substrates such as two vs. three dimensional substrates, growth on
beads, inside cylinders, or porous substrates.
[0095] Materials derived from early stage embryos, fetuses, or
adult tissues that may be tested include but are not limited to
acellular extracellular matrix prepared by the detergent extraction
of tissue from embryoid bodies, primitive endoderm, mesoderm, and
ectoderm, and the anlagen of differentiating organs and tissues or
living cells or tissues that when cocultured with the subject cells
cause an induction of differentiation.
[0096] Growth factors, adhesion factors, extracellular matrix
components, etc. may be tested individually or in various
combinations. In addition, these factors may be combined with
various culture conditions, e.g., vitamins and minerals, which may
also have an affect on the differentiation of stem cells. For
instance, it has been shown that oxygen tension may influence gene
expression and development in embryoid bodies. Bichet et al., 1999,
Oxygen tension modulates .beta.-globin switching in embryoid
bodies, FASEB J., 13: 285-95. In assay formats that expose test
cells to a variety of different combinations, care should be taken
to document the conditions applied to each sample so that results
may be correlated to the appropriate test conditions.
[0097] Growth factors and other compounds may be applied to stem
cells at about 0.1 to about 10 times their effective concentration;
for example, at about 2 times their effective concentration, for
varying periods of time, e.g. one hour to two months depending on
the timing of differentiation of the cell of interest during normal
development. Growth factors and other compounds can also be applied
repetitively or in a particular temporal order with other compounds
rather than simultaneously, with hours, days or weeks passing
between different administrations.
[0098] Screening Assays:
[0099] An embodiment of the present invention uses a screening
assay to identify agents or conditions that induce the
differentiation of totipotent, nearly totipotent, or pluripotent
stem cells, or cells therefrom; e.g., cells selected from the group
consisting of embryonic stem cells, embryonic germ cells, embryoid
bodies, ICM cells, morula-derived cells, non-ES stem cells, and
cells therefrom, and to characterize the type and degree of
differentiation that occurs in response to the agents or conditions
tested. For example, a screening assay of the invention can
comprise:
[0100] (a) separating individual totipotent, nearly totipotent, or
pluripotent stem cells, or cells therefrom, or groups of individual
cells, into one or a plurality of separate vessels which may be
open or closed, which vessels may be in the same or different
apparatus;
[0101] (b) isolating primary and/or progenitor cells from reference
tissues and placing said primary and/or progenitor cells into
separate vessels of a microarray thereby forming a control
reference library;
[0102] (c) exposing said separate vessels of totipotent, nearly
totipotent, or pluripotent stem cells, or cells therefrom, to the
same one or more putative differentiation-inducing compounds either
simultaneously or sequentially; and
[0103] (d) comparing said individual totipotent, nearly totipotent,
or pluripotent stem cells, or cells therefrom, or groups of cells,
to said reference library in order to evaluate the differentiation
of said individual cells or groups of cells.
[0104] High-Throughput Screening
[0105] A useful aspect of the present invention is that it provides
means for screening a large number of different types of stem
cells; e.g., a library of gene trap stem cells selected to have
gene trap markers that are activated when the stem cells are
induced to differentiate to a large number of different steps or
"nodes" in the complex, branching tree of possible differentiation
pathways leading to the partially or fully differentiated cell
types of an animal. Moreover, the present invention also provides
screening methods whereby one or more different types of stem cells
are exposed to a large number of different types of chemical and
biological agents and physical conditions, alone or in combination,
simultaneously or in various temporal combinations, to identify
sets of agents and conditions that induce the stem cells to
partially or fully differentiate into cell types of interest.
[0106] In performing the assays of the invention disclosed herein,
individual cells or individual groups of cells may be separated
into any type of array apparatus or assembly of compartments that
is convenient for systematically applying the test compounds and
evaluating differentiation. For instance, the cells may be
distributed into an apparatus comprising 10 to 100,000 different
vessels or compartments, or for some embodiments 100 to 100,000
compartments, or for others 1000 to 10,000 compartments, or
separate wells of one or more multi-well plates. The multi-well
plates that are used can have any number of wells; for example, the
screening assays of the invention can be performed using 24- or
96-well plates. In this embodiment, the reference library of
primary cells may be freshly isolated and distributed in a similar
array apparatus, or alternatively, frozen stock cells may be used.
In distributing the cells into compartments, e.g., the wells of one
or more 24- or 96-well plates, 1 to 10.sup.6 stem cells can be
added per cm.sup.2 of surface. For example, the screening assays of
the invention can be performed by adding 10 to 10.sup.5 cells per
cm.sup.2 of surface. Some ES cells require a minimum number of
cells to survive, for such cells, 3 to 10.sup.6 stem cells should
be added per cm.sup.2 of surface. Induction of differentiation by a
given set of conditions occurs with a statistical probability;
therefore, the more cells per well, the greater the likelihood that
a cell in the cell will be induced to differentiate.
[0107] Reference Library Cells and Cell Type-Associated Markers
[0108] The primary and/or progenitor cells used for the reference
library may include any cells of interest, i.e., any cells for
which the operator is interested in identifying differentiation
inducing compounds or compositions, including but not limited to
brain cells, heart cells, liver cells, skin cells, pancreatic
cells, blood cells, reproductive cells, nerve cells, sensory cells,
vascular cells, skeletal cells, immune cells, lung cells, muscle
cells, kidney cells, etc. The reference library cells are then used
as an experimental control when testing the exposed stem cells for
those that have differentiated into the particular primary cells in
the reference library. Functional assays specifically geared toward
detecting each of the cells in the reference library are performed
on the treated or exposed stem cells to correlate differentiation
with a particular cell type in the reference library.
[0109] For instance, depending on primary and secondary antibodies
and other ligand reagents available and what is known about the
molecular markers specific for particular cell types,
immunocytochemistry may be used to test treated stem cells for the
expression of proteins that correlate to specific cells in the
library. Alternatively, RT-PCR may be used to test the samples for
particular gene transcripts. There are many known molecular markers
of differentiation of cell types that are detectable, e.g. with
specific antibodies or by RT-PCR; examples include E-, P-, and
N-cadherins, keratin, chAT, tyrosine hydroxylase, gamma enolase,
PDX, amylase, CD34, VEGFR, cardiacmyosin, collagen 11, sex
determining region Y, frizzled-3, GATA 6, brachyury, PU.1 (Spi-1),
hepatocyte nuclear factor-3, alpha-2 type XI colagen, hepatic
lipase, nerve growth factor, sonic hedgehog, hematopoietically
expressed homeobox, enolase-2, keratin 19, osteoblast-specific
factor 2, globin transcription factor 1, myogenic factor 3, myosin
heavy polypeptide 2, dopamine transporter, CD34, human serum
albumin, pancreatin amylase, insulin promoter factor, beta-globin,
Oct 4, cardiac alpha-myosin heavy chain, cardiac myosin light chain
1, fibroblast growth factor 5 (FGF-5), SOX-1, alpha-fetoprotein
(AFP), EMX-2, engrailed-2 (En2), Hesx-1, Hox B1, Krox-20, Mush-1,
Nkx-1, Nkx-2, Pax-3, Pax-6, nestin, and GAPDH (a housekeeping gene,
useful as a control marker).
[0110] Cells in the reference library should be tested
simultaneously as a positive control, to ensure that a negative
result is not the failure of the assay itself rather than the
absence of the particular protein or transcript. Functional assays
could also be used to measure the production of enzymes or
metabolites produced by the particular reference primary and/or
progenitor cells, for instance by enzyme-linked immunosorbent
assays (ELISA), high performance liquid chromatography (HPLC),
Western blots, radioimmune assays, etc. For example, dopaminergic
neurons could be tested for KCl induced dopamine release, P-cells
for glucose dependent insulin release, cardiomyocytes for
synchronous contraction, hepatocytes for triacylglycerol
production, to name of few examples.
[0111] A second embodiment of the invention involves a method for
evaluating the differentiation of totipotent, nearly totipotent, or
pluripotent stem cells, or cells therefrom; in response to
different compounds or combinations of compounds, comprising:
[0112] (a) separating individual totipotent, nearly totipotent, or
pluripotent stem cells, or cells therefrom, or groups of individual
cells, into one or a plurality of separate vessels which may be
open or closed, which vessels may be in the same or different
apparatus;
[0113] (b) systematically exposing said separate vessels of
totipotent, nearly totipotent, or pluripotent stem cells, or cells
therefrom, to a panel of different putative
differentiation-inducing compounds or combinations thereof either
simultaneously or sequentially; and
[0114] (c) comparing said individual totipotent, nearly totipotent,
or pluripotent stem cells, or cells therefrom, or groups of cells,
to a reference differentiated or partially differentiated cell in
order to evaluate the differentiation of said individual cells or
groups of cells.
[0115] This embodiment differs from the first embodiment described
above in that the cells are treated with a panel of different
compounds and combinations of compounds, and the results are
compared with a single reference control in order to identify
particular conditions that resulted in directed differentiation
into that cell type.
[0116] Although any of the functional assays described above may be
used to analyze the results, this second embodiment is most
amenable to the use of RNA expression profiles. For instance,
expression profiles can be generated anytime at any pace and used
to form a library that catalogs the RNA expression profiles
according to what factors produced the specific profiles. Then, the
profiles may be compared at any time to expression profiles from
various reference primary cells in order to match each embryonic
differentiation profile with a primary cell. Such libraries may be
saved in electronic form, whereby matches in RNA expression
profiles as between the library members and any particular primary
or progenitor may be performed electronically rather than with the
naked eye.
[0117] A third embodiment involves a method for evaluating the
differentiation of totipotent, nearly totipotent, or pluripotent
stem cells, or cells therefrom, in response to different compounds
or combinations of compounds, comprising:
[0118] (a) isolating a transfected totipotent, nearly totipotent,
or pluripotent stem cell, or cell therefrom, wherein said cell is
transfected with at least one reporter gene, the expression of
which is operably linked to a promoter and/or gene of interest;
[0119] (b) expanding said transfected cell in culture;
[0120] (c) separating individual transfected cells or individual
groups of transfected cells into one or a plurality of separate
vessels which may be open or closed, which vessels may be in the
same or different apparatus;
[0121] (d) exposing said separate vessels of transfected cells to a
panel of different putative differentiation-inducing compounds or
combinations thereof either simultaneously or sequentially; and
[0122] (e) analyzing said individual transfected cells or groups of
cells in order to detect expression of said at least one reporter
gene.
[0123] Alternatively, this embodiment may be performed using gene
trap stem cells in which the marker DNAs are randomly inserted at
sites such that they are expressed upon activation of genetic loci
associated with the partial or complete differentiation of the stem
cells to a particular cell type. Such cells serve as functional
markers of differentiation, even when the genetic loci into which
the markers are inserted have not been identified.
[0124] In this embodiment, transfected totipotent, nearly
totipotent, or pluripotent stem cells, or cells therefrom, are
exposed to a panel of different compounds and combinations of
compounds, in order to identify those combinations that turn on
expression of a particular reporter gene construct.
[0125] Stem cells comprising a relevant reporter gene constructs
are known in the art as discussed supra, or alternatively, can be
produced according to known methods. For example, a reporter gene
may be targeted to the locus of a gene of interest, i.e., a gene
specifically expressed in the cell or tissue type desired, by
homologous recombination. By including an internal ribosome entry
sequence (IRES) and designing the vector such that insertion occurs
downstream of the endogenous stop codon, the transcript from the
targeted locus will act as a bicistronic message, making both the
endogenous protein and the protein encoded by the reporter gene. In
this manner, the targeted gene will not be functionally disrupted.
Alternatively, the targeted integration may be designed such that a
fusion transcript, and/or fusion protein results.
[0126] A second approach would be to construct reporter transgenes
using isolated promoter sequences for cell type specific genes.
This approach is not as sophisticated since homologous
recombination is not required, so it suffers from possible position
variegation in transgene expression. However, the constructs may be
made much more easily, and the use of good 5' and 3' flanking
sequences, and possibly insulator sequences, could alleviate some
of the variability.
[0127] The reporter gene strategy permits high-throughput and
non-invasive screening. Specifically, cells could be continuously
monitored, so the assay point would not be restricted to any
particular time period during the differentiation process. The
screening can be performed conducted by plating transgenic stem
cells onto 96 well plates, for instance, and supplying each well
with different conditions until reporter gene expression is
detected. This would enable different styles of experimental design
to rapidly be employed and evaluated. Further, this strategy could
also be coupled to other functional and morphological markers in
the same cell population.
[0128] Using the reporter gene strategy, the activation of gene
expression specific to certain cells types may be quantified with
respect to the purity of cells within the population. For example,
the methods of the invention could include the further steps
of:
[0129] (f) quantitatively determining the amount of detectable
signal; and
[0130] (g) comparing said amount of detectable signal with the
amount of signal produced by the same number of said transfected
cells in the absence of any test compound.
[0131] This aspect could also facilitate development of compound
combinations that yield purer cell populations. In addition, cells
expressing a reporter gene such as green fluorescence protein (GFP)
may be purified from other cells or undifferentiated cells in the
same sample by fluorescence activated cell sorting. Odorico et al.,
2001, Multilineage differentiation from human embryonic stem cell
lines, Stem Cells 19(3): 193-204.
[0132] Possible loci to be targeted for clinical applications are:
insulin in .beta.-cells, DOPA decarboxylase in dopaminergic
neurons, cardiac .alpha.-actin in cardiomyocytes, and albumin in
hepatocytes. Expression of these proteins are absolutely restricted
to the corresponding cell types, thus should provide a reliable
indicator or promoter source for the type of cells being
produced.
[0133] Reporter Genes Useful as Markers:
[0134] Reporter genes useful for the present invention encode
proteins that are detectable by any means, i.e., those that are
detectable by the naked eye or after microscopic, photographic or
radiographic analysis, or after contacting said exposed cells with
a reagent selected from the group consisting of chromogenic
substrates, dyes, sugars, antibodies, ligands, primers, etc.
Suitable reporter genes may encode polypeptides including but not
limited to green fluoresent protein (GFP), enhanced green
fluoresent protein (EGFP), luciferase, chloramphenical
acetyltransferase, .beta.-glucuronidase, .beta.-galactosidase,
neomycin phosphotransferase, alkaline phosphatase, guanine xanthine
phosphoribosyltransferase or .beta.-lactamase. See, e.g., U.S. Pat.
No. 5,928,88, herein incorporated by reference. The use of a marker
gene encoding a fluorescent protein such as GFP permits detection
of expression of the marker gene without injuring the cells.
Fluorogenic substrates include but are not limited to fluorescein
di-.beta.-D-galactopyranoside, resorufin
.beta.-D-galactopyranoside, DDAO galactoside, methylumbelliferyl
galactoside or its di-fluorinated analog, carboxyumbelliferyl
galactoside, fuorescent glycolipids, Amplex Red Galactose, PFB
Aminofluorescein, chloromethyl and lipophilic derivatives of
DiFMUG, 4-methylumbelliferyl .beta.-D-glucuronide, fluorescein di
.beta.-D-glucuronide, 5-(pentafluorobenzoylamino)fluorescein di
.beta.-D-glucuronide, DDAO .beta.-D-glucuronide, etc. Those skilled
in the art are familiar with many reagents for detecting
glycosidase activity.
[0135] A fourth embodiment involves a method for evaluating the
differentiation of transfected totipotent, nearly totipotent, or
pluripotent stem cells, or cells therefrom, in response to one or
more compounds, comprising:
[0136] (a) obtaining a library of transfected totipotent, nearly
totipotent, or pluripotent stem cells, or cells therefrom, each
transfected with at least one reporter gene, the expression of
which is operably linked to a pre-characterized promoter and/or
gene of interest;
[0137] (b) separating individual members of said library into one
or a plurality of separate vessels which may be open or closed,
which vessels may be in the same or different apparatus;
[0138] (c) exposing said separate vessels of transfected cells to
the same one or more putative differentiation-inducing compounds
either simultaneously or sequentially; and
[0139] (d) analyzing said individual members of said library in
order to detect expression of said at least one reporter gene.
[0140] This embodiment differs from the third embodiment described
above in that a library of different transfected cells, each
comprising a different reporter construct is exposed to a single
test compound or test combination (rather than a panel of compounds
being applied to a single type of stem cell representing a single
reporter construct).
[0141] As for the previous embodiment, this embodiment may be
performed using gene trap stem cells in which the marker DNAs are
randomly inserted at sites such that they are expressed upon
activation of genetic loci associated with the partial or complete
differentiation of the stem cells to a particular cell type, as
such gene trap cells function as markers of differentiation, even
when the genetic loci in which they are is inserted are
unidentified.
[0142] The present invention also includes identifying agents
and/or conditions that induce stem cell differentiation, and then
genetically modifying stem cells to facilitate isolation of a
characterized population of the differentiated cells; e.g., to use
in therapeutic trials in animal experimental models. A non-limiting
example of how this can be done is to transfect the stem cells with
a marker DNA encoding a non-immunogenic cell surface antigen that
is inserted into a genetic locus that is specifically expressed in
the differentiated cells to be isolated. Known methods, e.g.,
methods employing homologous recombination, can be used to target
the marker DNA to the desired locus. When the genetically altered
stem cells has differentiated into the desired cell type, the
marker gene is expressed and the cells become tagged with the
surface antigen. Methods for isolating genetically modified stem
cells based on expression of a marker protein such as a surface
antigen are described in Gay (U.S. Pat. No. 5,639,618), the
contents of which are incorporated herein by reference. In an
embodiment of this aspect of the invention, an isolation marker is
inserted into a stem cell to be expressed when the cell has
differentiated into a precursor of several specific cell types of
interest. Additional isolation markers can be inserted into the
same cell for expression when the precursor cells terminally
differentiate into specific cell types. This permits isolation of
either the precursor cells, or the terminally differentiated cell
types. For example, an isolation marker could be inserted into the
locus of the nestin gene, a marker of neural precursor cells, to
permit isolation of neural precursor cells; and additional
isolation markers can be inserted into genetic loci that are
specifically expressed in neuronal cells, glial cells, and
astrocytes, to permit efficient isolation of these cells types
after induction of their differentiation from the neural precursor
cells.
[0143] The present invention further includes identifying agents
and/or conditions that induce stem cell differentiation, and then
genetically modifying stem cells to constitutively express a marker
gene that permits detection of differentiated cells derived from
the genetically modified stem cells following their administration
to an animal.
[0144] Accordingly, it is an embodiment of the invention to utilize
cells from a species wherein a marker gene is used to identify a
differentiated cell of interest, and to transfect these cells with
a constitutively expressed marker such as Green Fluorescent Protein
(GFP). Differentiated cells resulting from these embryonic cells
are useful in testing the efficacy and safety of cell in cell
therapy in animal or human models. Expression of the cell
type-associated marker demonstrates to the investigator that the
cell type of interest is present in the target tissue of interest,
and the constitutively expressed marker identifies the administered
cells against the background of the host cells of the animal into
which the cells being tested were administered.
[0145] This embodiment may be performed by specifically preparing
and characterizing a tailored panel of stem cells comprising a
specific set of reporter constructs according to the techniques
discussed above. Methods and materials for making and analyzing
reporter gene constructs in eukaryotic cells, commonly called gene
trap vectors, are known in the art and could be geared toward
specific stem cells of interest once appropriate vectors are
identified. See, e.g., U.S. Pat. No. 5,922,601, herein incorporated
by reference in its entirety; see also Salminen et al., 1998, Dev.
Dyn. 212(2): 326-33 and Stanford et al., 1998, Blood 92(12):
4622-31, each incorporated by reference in its entirety.
[0146] The members of any specially designed panel may be
pre-characterized or specifically designed to be representative of
a particular cell type or lineage. Procedures for preselecting and
precharacterizing specific gene trap lines are known in the art.
See Baker et al., 1997, Dev. Biol. 185(2); Thorey et al., 1998,
Mol. Cell. Biol. 18(5): 3081-88; and Bonaldo et al., 1998, Exp.
Cell Res. 244: 125-36, each of which is incorporated herein in
their entirety. Alternatively, a panel of gene trap stem cells
having random insertions may be accumulated, and the insertions
that respond to a given compound or combination of compounds may be
characterized subsequently to exposure and identification. For
instance, the location of the insertion may be identified by
molecular cloning following PCR of the flanking endogenous genetic
material, and by sequencing outward from the inserted construct
using well-established techniques. See, e.g., Gossler et al., 1989,
Science 244(4903): 463-5, incorporated herein in its entirety.
[0147] A pluripotent cell that is particularly preferred for use in
designing such a panel is the Cyno-1 cell line, a pluripotent
primate stem cell line isolated from parthenogenetically activated
oocytes from Cynomologous monkeys.
[0148] Screening with Pre-Existing ES Cell Gene Trap Libraries
[0149] In screening stem cells to determine agents and conditions
that induce their differentiation to particular cell types, it is
useful to use a gene trap stem cell library comprised of stem cells
in which the marker genes are inserted in genetic loci that are
normally activated when the cells is induced to differentiate, and
are under transcriptional control of the endogenous promoters of
the loci where they are inserted. This ensures that expression of
the marker genes is controlled by the same regulatory signals
(e.g., transcription factors and factors that alter chromatin
structure) as the endogenous promoters of the loci where they are
inserted.
[0150] As an alternative to preparing an entirely novel gene trap
library, an embodiment of the present invention employs any of the
murine ES cell gene trap libraries that are already known and
available in the art. See, e.g., Cecconi & Meyer, 2000, FEBS
Lefts 480: 63-71; see also Durick et al., 1999, Genome Res. 9(11):
1019-25. For instance, the German Gene Trap Consortium (GGTC) has
been established in Germany to provide a reference library of gene
trap sequence tags (GTST) in mouse embryonic stem cells. See Wiles
et al., 2000, Nature Genetics 24(1), incorporated by reference in
its entirety. Sequence information on the GTST library is
accessible at the Internet site of the GGTC, and the mutant ES
cells are freely available upon request to the scientific
community. Another library of gene trap murine ES cells, called
OmniBank.RTM., is also available from Lexicon Genetics, Inc. (The
Woodlands, Tex.), who have reportedly characterized over 20,000
sequence-tagged mutations. The OmniBank.RTM. database may be
searched using keywords or nucleotide or protein sequences via the
Internet site of Lexicon Genetics, Inc. See also Zambrowicz &
Friedrich, 1998, Int. J. Dev. Biol. 42(7): 1025-36; Zambrowicz et
al., 1998, Nature 392: 609-11; see also U.S. Pat. Nos. 6,080,576,
6,207,371 and 6,136,566, each herein incorporated by reference in
their entirety. Another group reported the successful recovery of
115 sequences from 153 cell lines using 5' RACE technology. Townley
et al., 1997, Genome Res. 7: 293-298, incorporated by reference in
its entirety. Sequence information from some of these murine ES
cell clones is available on the University of California/Berkeley
web site of the Skarnes lab. In addition, details on a large number
of other academic mouse ES cell tagging efforts have also recently
been reported. Chowdhury et al., 1997, Nucleic Acids Res. 25:
1531-1536; Hicks et al., 1997, Nat. Genet. 16: 338-344; Couldrey et
al., 1998, Dev. Dyn. 212: 284-292; and Voss et al., 1998, Dev. Dyn.
212: 171-180, each of which is incorporated by reference herein in
its entirety.
[0151] Gene-trap ES cells have been used to generate large numbers
of mutant organisms for genetic analysis. The retrieval of
transgenic mice made from gene trap ES cells has allowed for
trapped genes to be characterized and segregated based on tissue
expression profile, or subcellular expression characteristics. Some
predict that genome-wide gene-trapping strategies, which integrate
gene discovery and expression profiling, can be applied in a
parallel format to produce living assays for drug discovery. Durick
et al., 1999, supra. The use of gene trap clones in in vitro
studies, on the other hand, has been limited.
[0152] U.S. Pat. No. 6,080,576 to Zambrowicz suggests using gene
trap ES cells to screen for secreted molecules that induce
apoptosis or hematopoietic cell differentiation. However, this
approach is geared toward identifying insertions that cause
over-expression of endogenous genes, and does not provide a format
for systematically screening large numbers of compounds for their
effect on stem cell differentiation. Similarly, Russ and colleagues
disclose the identification of genes induced by factor deprivation
in hematopoietic cells undergoing apoptosis using gene trap
mutagenesis. Russ et al., 1996, Identification of genes induced by
factor deprivation in hematopoietic cells undergoing apoptosis
using gene-trap mutagenesis and site-specific recombination, Proc.
Natl. Acad. Sci. USA 93: 15279-84. However, this approach looks for
genes activated during programmed cell death rather than genes
activated during embryonic or stem cell differentiation.
[0153] Era and colleagues utilize a LacZ reporter gene similar to
that used in gene trap strategies in order to characterize
hematopoietic lineage-specific gene expression by ES cells in an in
vitro differentiation induction system. Era et al., 2000, Blood
95(3): 870-78. However, this approach was geared toward analyzing a
particular promoter of interest and determining which section of
the promoter was responsible for differentiation-induced
expression. There was no suggestion to use the promoter constructs
to screen for growth factors or other compounds that are involved
in particular cell lineage differentiation pathways.
[0154] Bonaldo and colleagues used gene-trap and pre-selection
analysis of isolated cell lines to identify fusions that are
expressed during embryonic development in response to specific,
single growth factors. They do not use the cells identified,
however, to screen for combinations of factors that direct the
development of those cells. In fact, the low-serum medium employed
in the screening process was only suitable for short term screening
lasting about 24 hours. See Bonaldo et al., 1998, supra. Thus,
Bonaldo et al. presents a means of preselecting and
precharacterizing cells containing fusions in the early developing
embryo, but does not disclose the use of such cells in screening
for factors that direct the differentiation of specific cells and
tissues.
[0155] Similarly, Forrester and colleagues used gene-trap
technology to identify genes specifically expressed in response to
retinoic acid during embryogenesis. Forrester et al., 1996, An
induction gene trap screen in embryonic stem cells: Identification
of genes that respond to retinoic acid in vitro, Proc. Natl. Acad.
Sci USA 93: 1677-82. However, they also did not use such cells to
screen for growth factor combinations that direct the development
of specific cells and tissues.
[0156] Thus, this is the first disclosure of which the present
inventors are aware, that proposes to use the gene trap ES cell
libraries as a tool for screening growth factors, adhesion factors,
extracellular matrix materials, etc., for compounds and
combinations that mediate the directed differentiation of stem
cells. The ES cells identified as corresponding to a specific
combination of growth factors may be used to make transgenic
embryos or animals, in order to correlate in vivo temporal and
spatial gene expression with the in vitro data obtained in the
disclosed method.
[0157] As indicated by the foregoing description, libraries of
totipotent murine and non-human gene trap stem cells can be
assembled from existing cell lines, or novel libraries can be made
using known techniques. When gene trap marker DNAs are inserted
randomly, developing or mature animals cloned from the gene trap ES
cells can be sacrificed and analyzed histologically to identify the
gene trap stem cell lines that contain markers that are activated
in particular cell types for use in the screening assays of the
invention.
[0158] Alternatively, gene trap marker DNAs can be inserted into ES
or EG cells, either directly or by deriving genetically modified ES
or EG cells from a nuclear transfer embryo produced with a
genetically modified nuclear donor cell. For example, a library of
totipotent human gene trap stem cells can be produced by deriving a
set of genetically modified human ES or EG cells from nuclear
transfer embryos produced with genetically modified nuclear donor
cells. The gene trap ES or EG cells are then expanded and used to
produce embryoids containing diverse types of differentiated cells.
Histological analysis is performed to identify the gene trap stem
cell lines that contain markers that are activated in particular
cell types for use in the screening assays of the invention.
Alternatively, the totipotent cells can be injected into an animal
to produce teratomas containing diverse types of differentiated
cells, and histological analysis of these performed to identify the
gene trap stem cell lines that contain useful markers of
differentiation.
[0159] The present invention has broad applications. For example,
in addition to identifying agents and conditions that induce and
direct differentiation, the screening methods of the present
application permit identification of agents and conditions that
promote cell survival (survival factors), and of agents and
conditions that promote mitogenesis (mitogenic factors). For
example, cells are cultured for a period of 1-14 days with exposure
to a panel of different agents and conditions that are putative
survival and/or mitogenic factors, and the effects of the various
treatments on cell survival and/or cell proliferation over the time
interval of the assay is determined. Agents and conditions that
decrease the loss or death of particular cell types can be detected
by the assay in this manner, and may be regarded as survival
factors. Similarly, agents and conditions that increase cell
proliferation over the course of the assay are mitogenic factors.
The combination of information regarding differentiation, survival,
and mitogenic factors is useful in identifying and optimizing
conditions that are useful for producing desired quantities of
medically useful cell types.
[0160] In another aspect, the invention encompasses compositions
and formulations comprising the compounds and compositions
identified using the disclosed methods, and the use thereof to
direct the development of cells and tissues from totipotent, nearly
totipotent, and pluripotent stem cells, and cells therefrom, to
isolate cells and tissues for use in treatments and transplantation
therapies. In particular, the identified combinations of factors
may be used to induce the differentiation of cells on polymeric
matrices, i.e., as disclosed in U.S. Pat. Nos. 6,214,369,
6,197,575, and 6,123,727, each of which is herein incorporated by
reference in its entirety.
[0161] The combinations identified by the disclosed methods may
also be used to induce the production of different types of cells,
either separately or in conjunction, in order to design and recover
tissues and/or artificial organs constituted of different cell
types.
[0162] Other embodiments, variations and modifications of the
assays and methods disclosed herein will be envisioned by those in
the art upon reading the present disclosure, and should also be
included as part of the invention.
EXAMPLE 1
[0163] Conditioning Totipotent Stem Cells to Grow and Maintain an
Undifferentiated State in the Absence of Feeder Cells:
[0164] ES-like cells derived from the inner cell mass of
parthenogenetic Cynomologous monkey embryos, Cyno-1 were originally
cultured on mitotically inactivated mouse embryonic fibroblast
derived from D12 fetuses (strain 129).
[0165] The culture media was:
1 DMEM (High Glucose) (Gibco #11960-044) 425 ml Fetal Calf Serum
(Hyclone) 75 ml MEM non essential AA x100 (Gibco #11140-050) 5 ml
L-Glutamine 4 mM 2-mercatoethanol (Gibco #21985-023) 1.4 ml
[0166] The cells were passaged mechanically every 4 to 5 days.
[0167] To condition the cells to grow in the absence of feeder
cells to improve the screening assay, the cells were passaged
mechanically into a non-coated Polystyrene cell culture plate
(Corning)
[0168] For the first two days, cells were cultured in conditioned
media from the original cultures (described above)
[0169] On day three, conditioned media was replaced by:
[0170] Human Endothelial-SFM Basal Growth Medium (Gibco #11111-044)
500 ml
[0171] EGF-Human Recombinant (Gibco #10458-016) 10 .mu.g
[0172] bFGF (Gibco #13256-029) 10 .mu.g
[0173] Human Plasma Fibronectin (Gibco #33016-023) 1 mg
[0174] The colonies maintained their pluripotent phenotype
(morphology and AP staining) for up to one week. The cultures
appeared that grew in the absence of feeder fibroblasts while
maintaining an undifferentiated state. This new line designated
Cyno-1 FF displays the morphology of undifferentiated ES-like cells
in that they have small cytoplasmic to nuclear ratios, prominent
nucleoli, and are alkaline phosphatase positive (FIG. 1).
EXAMPLE 2
[0175] Screen Using Primate ES-Like Cells and Analysis by
Microscopy and RT-PCR:
[0176] Approximately 10.sup.5 ES-like stem cells from
parthenogenetic monkey embryos (Cyno-1 FF cell line, see Example 1)
were plated in duplicate 24 well plates in the presence of mouse
embryonic fibroblast-conditioned medium for two days. The media was
then aspirated and replaced with DMEM medium with 15% fetal bovine
serum, added nonessential amino acids, 5.times.10.sup.-5 M
.beta.-mercaptoethanol, 2 mM L-glutamine, 100 .mu.g/ml penicillin,
and 100 .mu.g/ml streptomycin. The cells were then cultured in the
presence of growth factors or cytokines in order to direct their
differentiation. Working stock solutions of the cytokines were
prepared in 0.1% bovine serum albumin (BSA) in phosphate-buffered
saline (PBS). Diluted cytokines were applied on Day 0. To each
well, 7.5 .mu.l of diluted factor was added from the working stock
solutions to obtain the following final concentrations:
[0177] VEGF-A (165 kDa) (R&D Biosystems cat# 293VE) was used at
20 ng/mL,
[0178] LAP (R&D #246-LP) at 50 ng/mL,
[0179] Flt-3/Flk-2 ligand (R&D #308-FK) at 5 ng/mL,
[0180] TGF beta-1 (R&D #240-B) at 0.1 ng/mL,
[0181] IGF-1 (R&D #291-G1) at 10 ng/mL,
[0182] PIGF (R&D #264-PG) at 20 ng/mL,
[0183] Tie-1/Fc chimera (R&D #619-TI) at 100 ng/mL,
[0184] BMP-2 (R&D #355-BM) at 500 ng/mL,
[0185] BMP-4 (R&D #314-BP) at 250 ng/mL,
[0186] BMP-5 (R&D #615-BM) at 2 .mu.g/mL,
[0187] FGF-17 (R&D #319-FG) at 50 ng/mL,
[0188] TGF-alpha (R&D #239-A) at 0.5 ng/mL,
[0189] Fibronectin (human 120 chymotryptic fragment, Gibco
#12159-018) at 50 ng/mL,
[0190] Merosin (Gibco #12162-012) at 50 ng/mL,
[0191] Tenascin (Gibco #12175-014) at 50 ng/ml,
[0192] IL-1-alpha (R&D # 200-LA) at 10 .mu.g/mL,
[0193] FGF-4 (R&D #235-F4) at 0.25 ng/mL,
[0194] SCF (R&D #255-SC) at 10 ng/mL,
[0195] bFGF (R&D #233-FB) at 1.0 ng/mL,
[0196] PDGF (R&D #120-HD) at 5.0 ng/mL,
[0197] PECAM-1 (R&D #ADP6) at 1.0 .mu.g/mL,
[0198] anti-FGF-4 antibody (R&D #AF235) at 0.5 .mu.g/mL,
[0199] anti-Cripto-1 antibody (R&D #AF145) at 0.5 .mu.g/mL,
[0200] and a control of the same volume of 0.1% BSA in PBS.
[0201] To coat a well with an ECM component, a solution of the ECM
component at a concentration of 10 .mu.g/mL in PBS was added to the
well to be coated and incubated for at least one hour, and then
removed by aspiration.
[0202] The plates were cultured at 37 deg. C. at atmospheric
O.sub.2 and 5% CO.sub.2, one for three and one for ten days. Table
1 in FIG. 2 identifies the factors that were added to each of the
wells of duplicate 24-well plates. One plate was harvested on Day
3, and the other plate was harvested on Day 10. Analysis by phase
contrast microscopy and RT-PCR revealed many unique differentiated
cell types, as discussed below.
[0203] Analysis of Cell Morphologies by Phase Contrast
Microscopy:
[0204] Following exposure to Flt-3 ligand, the Cyno-1 FF ES-like
cells differentiated into cells that appeared to be vascular
endothelial cells (derivatives of mesodermal differentiation).
Cells having the appearance of vascular endothelial cells were
observed by five days in the wells with added Flt-3 ligand, and
were more evident in these wells by day 11. FIG. 3 is a photograph
of primate Cyno-1 FF cells exposed to Flt-3 ligand.
[0205] Exposed to TGF beta-1 induced Cyno-1FF cells to acquire
morphologies that appeared to be those of mesodermal and neural
stem cells. FIG. 4 shows mesoderm and cells with the morphology of
nestin positive neuronal stem cells obtained by the culture of
Cyno-1 FF cells in the presence of TGF beta-1.
[0206] Cyno-1 FF cells cultured in the presence of the
extracellular matrix protein tenascin induced the formation of a
distinctive population of cells that had the appearance of
endodermal precursor cells. The appearance of the cells in the
presence of tenascin was strikingly different from that of the
cells in the control well. This result indicates that different
concentrations of this particular extracellular matrix component
and/or its removal or inactivation can be used to direct the
differentiation of totipotent and pluripotent stem cells. FIG. 5
shows cells with the appearance of endodermal precursor cells
obtained by the culture of Cyno-1 FF cells in the presence of the
extracellular matrix protein tenascin.
[0207] Cyno-1 FF cells that were exposed to other putative
differentiation-inducing agents in other wells of the assay plate
were also induced to differentiate to have distinctive morphologies
and to express cell type-associated genes. For example, FIG. 6
shows the appearance of cells cultured in the presence of Tie-1
receptor/Fc chimera. Cells cultured in the presence of BMP-2
acquired the morphology and appearance of connective tissue
fibroblast-like cells, as shown in FIG. 7.
[0208] RT-PCR Analysis of Expression of Cell Type-Associated
Genes:
[0209] The expression of cell type-associated genes by the Cyno-1
FF cells exposed to the panel of putative differentiation-inducing
agents shown in FIG. 2 was assayed by RT-PCR using the following
standard protocols.
[0210] (a) RNA was harvested from cells using kit: Ultraspec-II
RNA, Item No. BL-12050 (Bioflex Labs, Inc) and included
protocol.
[0211] (b) The isolated RNA was amplified using listed primers and
kit: Enhanced Avian RT First Strand Synthesis kit, Item No. STR-1
(Sigma-Aldrich, Inc)
[0212] (c) The amplified RNA was harvested from cells and stored at
-70.degree. C. in ethanol.
[0213] (d) Reverse transcription reaction:
[0214] RNA was resuspended to 30 ul, and the following reagents
were added:
[0215] 2 ul dNTP mixture
[0216] 2 ul Random nonamers
[0217] the mixture was heated to 80.degree. C. for 12 minutes, then
transferred to an ice bath for 5 minutes
[0218] the following reagents were added:
[0219] 4 ul 10.times.RT buffer
[0220] 1 ul RNAse inhibitor
[0221] 2 ul reverse transcriptase
[0222] the reaction was then thermocycled using the following
conditions:
[0223] 24.degree.-15 min
[0224] 42.degree.-50 min
[0225] 95.degree.-30 sec
[0226] 4.degree.-hold
[0227] the mixture was then aliquotted with 3 ul/sample and was
stored at -70.degree. C. until use.
[0228] (e) Polymerase chain reaction:
[0229] The following reagents were added to each sample:
[0230] 2 ul primer pair mix (50 pmol/ul)
[0231] 5 ul MgCl2
[0232] PCR reaction mixture (for each sample):
[0233] 5 ul 10.times. buffer (without Mg)
[0234] 4 ul dNTP mix (10 mM)
[0235] 0.5 ul Taq (Sigma)
[0236] 0.055 ul HotStart Taq (Qiagen)
[0237] 30.5 ul H20
[0238] the reaction was then thermocycled for 35 cycles using the
following conditions:
[0239] 94.degree. C.-2 min
[0240] 94.degree. C.-30 sec
[0241] 45.degree. C.-1 min
[0242] 72.degree. C.-2 min
[0243] 72.degree. C.-10 min
[0244] 4.degree. C.-hold
[0245] The primers that were used to detect expression of cell
type-associated genes by RT-PCR, and the expected sizes of the
products, are shown in Table 2 shown in FIG. 8. The PCR products
were visualized by polyacrylamide gel electrophoresis, ethidium
bromide staining, and illumination with uv light. The bands were
identified by predicted size and relative intensity determined by
comparison with GAPDH intensity.
[0246] Examples of the results, demonstrating detection of specific
differentiation pathways in the endoderm, mesoderm, and ectoderm
germ layers in the wells by RT-PCR, is shown in FIG. 9.
[0247] FIG. 9 shows that Cyno-1 FF cells induced to differentiate
by different differentiation-inducing agents express different but
sometimes overlapping combinations of cell type-associated genes.
For example, cells exposed to VEGF-A expressed ChAT, keratin-19,
and nestin, and cells exposed to tenascin expressed ChAT, nestin,
and GATA-4. The strongest induction of ChAT (choline
acetyltransferase) and therefore, of neuronal differentiation was
seen in well 10-14 in the presence of the extracellular matrix
component tenascin, and in well 10-15 in the presence BMP-5. ChAT
was also induced by TGF-beta-1, IGF-1, FGF-4, bFGF, tenascin, and
anti-Cripto-1 antibody. The best endothelial/hematopoietic
conditions observed were in the presence of Flt-3 ligand. This
correlated well with the endothelial morphology observed by phase
contrast shown in FIG. 3. Interestingly, the best conditions
observed to induce endothelial differentiation were also in the
presence of the extracellular matrix component tenascin.
[0248] In contrast to the results obtained with cells cultured in
wells containing differentiation-inducing agents as described
above, expression of cell type-associated genes by control Cyno-1
FF cells cultured in medium without the added putative
differentiation-inducing agents was no detected by the RT-PCR
assay. This result is evidence that the above-described assay
detected genuine differentiation-inducing effects.
EXAMPLE 3
[0249] Screen Using Primate ES-Like Cells and Analysis by
Immunocytochemistry:
[0250] The presence of products of the expression of cell
type-associated genes in Cyno-1 FF cells exposed to putative
differentiation-inducing agents in one of the 24 well plates
prepared according to Example 2 was detected by immunocytochemistry
(ICC).
[0251] Solutions for Immunocytochemistry:
[0252] Fixative: 4% Paraformaldehyde
[0253] Permeabilization Solution: DPBS+1% TritonX-100
[0254] Blocking Solution: DPBS+150 mM glycine+3 mg/ml BSA
[0255] Rinsing Solution: DPBS+0.1% Triton X-100
[0256] Antibody Diluent: DPBS+0.1% Triton X-100+3 mg/ml BSA
[0257] General Protocol for Immunocytochemistry:
[0258] Rinse cells in DPBS (with Ca/Mg so cells do not dissociate)
3.times..
[0259] Add 4% Paraformaldehyde, Incubate at RT for 20-30 min.
[0260] Remove fixative with a Pasteur pipette and wash 3.times.
with PBS. At this point cells can be stored at 4C for long periods
of time if wrapped in parafilm.
[0261] Add blocking solution and incubate at RT for at least 1 hour
(this can be prolonged as long as overnight).
[0262] Remove blocking solution and replace with primary antibody
(diluted . . . generally dilutions of 1:10 to 1:100 work well).
[0263] Incubate at RT for at least 1 hour.
[0264] Remove primary antibody and wash 3.times. with PBS over 45
minutes.
[0265] Add secondary antibody (diluted . . . generally dilutions of
1:50 to 1:500 work well).
[0266] Rinse 3.times. in PBS over 45 minutes. Add 5 ug/ml Hoechst
or DAPI to first rinse.
[0267] Sample is ready for imaging.
[0268] Antibodies Used:
[0269] GATA-4: Item # sc-1237 (Santa Cruz Biotechnology, Inc.)
[0270] Goat IgG used at dilution of 1:75
[0271] Nestin: Item# 611659 (BD Transduction Laboratories, Inc)
[0272] Mouse IgG1 used at dilution of 1:75
[0273] Desmin: Item# D-1033 (Sigma-Aldrich, Inc)
[0274] Mouse IgG1 used at dilution of 1:20
[0275] Goat anti-Mouse IgG--FITC conjugate: Item# F-0257
(Sigma-Aldrich, Inc)
[0276] Used at dilution of 1:50
[0277] Mouse anti-Goat IgG--FITC conjugate: Item#sc-2356 (Santa
Cruz Biotechnology, Inc.)
[0278] Used at dilution of 1:50
[0279] The ICC assay successfully detected gene expression products
associated with each of the three embryonic germ layers. FIG. 10
demonstrates the detection by ICC of desmin, a marker for mesoderm,
and FIG. 11 demonstrates the detection of nestin, primarily a
marker for ectoderm, but sometimes of endoderm, in Cyno-1 FF cells
exposed to differentiation-inducing agents. The expression of
GATA-4, a marker for endoderm, was also detected by ICC in Cyno-1
FF cells exposed to differentiation-inducing agents (results not
shown).
EXAMPLE 4
[0280] Screen Using Primate ES Cells, Induction of Differentiation
by Physical Conditions:
[0281] Cyno-1 FF ES-like cells were plated in wells of a 24 well
plate as described in Example 2, and were incubated under low
oxygen partial pressure (5%). A control plate of the same cells was
incubated in ambient oxygen. Analysis of cellular morphologies
showed that the cells incubated under low oxygen partial pressure
(5%) were induced to acquire different morphologies than the
control cells incubated under ambient oxygen. This example
demonstrates the importance of screening various physical as well
as chemical factors to identify conditions or factors that induce
differentiation of stem cells into desired cell types.
EXAMPLE 5
[0282] Screen for Agents that Induce Differentiation of Murine ES
Cells into Myocardial Cells:
[0283] Approximately 20,000 murine ES cells (strain J1) were plated
in a 24 well tissue culture plate without feeder fibroblasts or LIF
in 1.5 mL of DMEM Medium with 15% fetal bovine serum, added
nonessential amino acids, 5.times.10.sup.-5 M 2-mercaptoethanol, 2
mm L-glutamine, 100 ug/ml penicillin, and 100 ug/ml streptomycin.
The cells were incubated and allowed to differentiate in the
presence of the same added factors and in the same manner as
described in Example 2. After ten days of differentiation, the
morphologies of the cells were examined by phase contrast
microscopy to detect rhythmically contracting cells as evidence of
myocardial differentiation. Only one well, well #16 containing
IL-1-alpha, contained contracting rhythmically myocardial cells.
Interestingly, these cells were and consistently found to be
growing in association with underlying endothelial cells. FIGS. 12A
and 12B are phase contrast photographs of the cells in well #16.
The arrowhead in the figure on the left (FIG. 12A) points to a
beating myocardial cell. The arrowhead in the figure on the right
(FIG. 12B) points to an endothelial cell inducers adjacent to
myocardial cells.
EXAMPLE 6
[0284] Screen for Agents that Induce Differentiation of Murine ES
Cells; Detection by ICC:
[0285] Approximately 5,000 murine ES cells (strain J1) were plated
in a 24 well tissue culture plate without LIF in 1.5 mL of DMEM
Medium with 15% fetal bovine serum, added nonessential amino acids,
5.times.10.sup.-5 M 2-mercaptoethanol, 2 mM L-glutamine, 100 ug/ml
penicillin, and 100 ug/ml streptomycin. The cells were allowed to
differentiate in the presence of FGF-4 and/or TGF-beta-1
(concentrations as in Example 2), in the presence or absence of
inducer fibroblasts, or in the presence or absence of type I
collagen and human plasma fibronectin (the wells were precoated by
incubating for an hour with 10 ug/mL of the ECM proteins, and then
removing and rinsing in PBS). The combinations of putative
differentiation-inducing agents in each well are shown in Table 3
of FIG. 13.
[0286] After incubating the cells for five days in the presence of
the putative differentiation-inducing agents, the cells in the
wells were assayed for expression of cell type-associated genes by
ICC. Primary antibodies to desmin, nestin, and GATA-4 were applied
to the cells and visualized by fluorescence microscopy as described
in Example 3 above. FIG. 14 shows immunofluorescence from
anti-desmin antibody bound to desmin, a marker of mesodermal cell
lineages, in murine ES cells cultured in TGF-beta-1 and FGF4 for
five days on type I collagen and human plasma fibronectin.
[0287] The expression of cell type-associated genes such as GATA-4
and nestin by the murine ES cells in the wells that were induced to
differentiate was also detected by RT-PCR assay performed as
described in Example 2 (data not shown).
EXAMPLE 7
[0288] Screen with Murine Gene-Trap ES Cell Lines; Detection by
X-Gal Staining and ICC:
[0289] Cells of the murine gene trap ES cell lines K18E2 and M7H7
each have DNA encoding beta-galactosidase inserted as a marker gene
in a genetic locus that is activated when the cells differentiate.
The DNA encoding beta-galactosidase is inserted in-frame with
correct orientation at a site such that it is expressed and
beta-galactosidase is produced when the genetic locus in which it
is inserted is activated. Accordingly, the beta-galactosidase
coding sequence operates as a marker permitting detection of the
differentiation of K18E2 and M7H7 ES cells. The beta-galactosidase
marker DNA is inserted at different loci in K18E2 and M7H7 ES
cells, and the sets of conditions that leads to activation of the
marker gene are not the same for the two cell types. The
beta-galactosidase marker in K18E2 ES cells is expressed in many
early differentiated cell lineages; the beta-galactosidase marker
in M7H7 cells is expressed in early mesoderm and retains expression
in endothelial and hematopoietic pathways.
[0290] Cells of murine gene trap cell lines K18E2 were treated as
described in Example 6 above and subsequently stained with X-gal to
detect expression of the marker beta-galactosidase gene. X-gal
staining is generally well known in the art. Briefly, the cells
were washed once with 0.1 M phosphate buffer, fixed at room
temperature in 25% gluteraldehyde in 0.1 M phosphate buffer, washed
again five times in phosphate buffer, and stained overnight at 37
degrees C. with X-gal stain. The pH of the buffer was in the range
of 7.0-8.0 depending on the cells used.
[0291] FIG. 15 shows the detection of X-gal staining of K18E2 ES
cells that were cultured for five days on type I collagen and human
plasma fibronectin in the presence of TGF-beta-1 and FGF-4.
Detection of expression of the beta-galactosidase marker gene in
cells derived from the K18E2 ES cells indicates that the cells were
induced to differentiate.
[0292] Expression of the beta-galactosidase marker gene in K18E2
and M7H7 ES cells that were cultured in the presence of
differentiation-inducing agents was also detected by ICC. FIG. 16
shows the detection of beta-galactosidase by ICC (using antibody to
beta-galactosidase) in M7H7 ES cells that were cultured for five
days on type I collagen and human plasma fibronectin in the
presence of TGF-beta-1 and FGF-4. Cell nuclei were co-visualized by
DAPI staining. FIG. 17 shows the detection of beta-galactosidase by
ICC in K18E2 ES cells that were cultured for five days on type I
collagen and human plasma fibronectin in the presence of FGF-4.
[0293] Using RT-PCR to detect expression, the beta-galactosidase
marker gene in murine gene trap ES cells was also shown to be
activated when the cells were induced to differentiate by other
cells (data not shown).
EXAMPLE 8
[0294] Screen for Induction of Differentiation by Cell-Cell
Interactions:
[0295] Murine gene trap K18E2 and M7H7 ES cells were plated in
wells of a 24-well tissue culture plate (5,000 to 20,000
cells/well) and were allowed to differentiate in the presence of
FGF-4 and TGF-beta-1, generally as described in Example 6 above,
except that in some of the wells, the cells were plated onto a
layer fibroblast mesenchymal inducer cells. After incubation for
five days, the cells were all transferred to wells containing FGF-4
and TGF-beta-1 without inducer cells, and were cultured for an
additional five, days. Following this treatment, expression of the
beta-galactosidase marker gene was detected by the ICC protocol
described in Example 3. The images in FIGS. 18-21 are of labeling
using monoclonal anti-.beta.-galactosidase (G-6282 Sigma-Aldrich,
Inc.) primary antibody and anti-mouse IgM FITC conjugated (F9259
Sigma-Aldrich, Inc.) secondary antibody.
[0296] Results:
[0297] FIG. 18 shows the presence of .beta.-galactosidase in K18E2
cells that were cultured with FGF-4 and TGF-.beta.1 on inducer
fibroblasts for 5 days, then sub-cultured for an additional 5 days
with FGF-4 and TGF-.beta.1 alone. FIG. 19 shows the presence of
.beta.-galactosidase in M7H7 cells that were cultured with FGF-4
and TGF-.beta.1 on inducer fibroblasts for 5 days, then
sub-cultured for an additional 5 days with FGF-4 and TGF-.beta.1
alone. FIG. 20 shows the presence of .beta.-galactosidase in K18E2
cells that were cultured with FGF-4 and TGF-.beta.1 in the absence
of inducer fibroblasts, and then sub-cultured for 5 more days in
same conditions. FIG. 21 shows the presence of .beta.-galactosidase
in M7H7 cells that were cultured with FGF-4 and TGF-.beta.1 in the
absence of inducer fibroblasts, and then sub-cultured for 5 more
days in same conditions.
[0298] The beta-galactosidase marker gene was expressed by both
lines of gene trap stem cells cultured with FGF-4 and TGF-.beta.1
on inducer fibroblasts, and also by the same stem cells cultured
with FGF-4 and TGF-.beta.1 alone. However, the beta-galactosidase
marker gene was expressed by the M7H7 cells cultured with FGF-4 and
TGF-.beta.1 on inducer fibroblasts significantly more strongly than
by the M7H7 cells that were cultured with FGF-4 and TGF-.beta.1
alone. The beta-galactosidase marker gene of M7H7 is activated when
the cells are induced to differentiate into cells of the mesodermal
lineage, and are therefore useful for identifying conditions that
induce the stem cells to differentiate into hematopoietic cells.
This example demonstrates the use of the invention to identify
cell-cell interactions between stem cells and inducer fibroblasts
that operate to induce differentiation of stem cells into cells of
the mesodermal lineage, e.g., for producing hematopoietic
cells.
EXAMPLE 9
[0299] Directing Differentiation with Multi-Nodal Markers:
[0300] This example demonstrates how multi-nodal markers can be
used to identify differentiated cell types.
[0301] Cell line A is a totipotent gene trap stem cell line with a
gene trap marker that is expressed when the cells are exposed to
three different sets of conditions that direct them to
differentiate, respectively, into heart, lung, and kidney.
[0302] Cell line B is a totipotent gene trap stem cell line with a
different gene trap marker that is expressed when the cells are
exposed to three different sets of conditions that direct them to
differentiate, respectively, into lung, brain, and eye.
Cell Types in Which the Gene Trap Marker is Expressed:
[0303]
2 Cell line A Cell line B heart eye lung lung kidney brain
[0304] Screening is performed to identify a set of conditions that
activates the marker in both cell lines; this set of conditions is
expected to direct differentiation to lung.
EXAMPLE 10
[0305] Screening in Eggs:
[0306] An array of avian eggs is used as the set of compartments in
which screening for differentiation is performed. 10.sup.2 to
10.sup.5 totipotent, nearly totipotent, or pluripotent stem cells;
e.g., murine or primate ES cells, are introduced into each egg. One
or more putative differentiation-inducing agents; e.g., growth
factors, cytokines, ECM compounds, and/or inducer cells, are then
added to the cells in each egg in various combinations and temporal
sequences. The eggs are incubated and activation of cell
type-associated genes in the cells is detected by RT-PCR.
[0307] The assay can be performed using gene trap ES cells having
gene trap markers that are activated when the stem cells
differentiate into specific cell types. Use of such cells permits
two types of screening to be performed. In one, an array of eggs is
prepared with each egg containing the same type of gene trap stem
cell, and a different combination of putative
differentiation-inducing agents. In the other, an array of eggs is
prepared with each egg containing stem cells having a different
gene trap marker that is activated when the cell is induced to
differentiate, and the same combination of putative
differentiation-inducing agents. The first assay is a screen to
identify agents or conditions that direct differentiation of stem
cells into a specific cell type. The second assay identifies cell
type-associated markers that are activated by a particular set of
putative differentiation-inducing agents.
EXAMPLE 11
[0308] Screens Utilizing Lineage Tracers Introduced by
Site-Specific Recombination:
[0309] For efficient detection of the activation of a genetic locus
that is only transiently activated at a step or "node" in the
branching pathway leading to differentation to a desired specific
cell type, gene trap stem cells can be made by inserting two coding
sequences into the genome of the stem cell:
[0310] (i) a sequence encoding a recombinase that is inserted into
the locus in-frame with correct orientation at a site such that it
is expressed and recombinase is produced when the genetic locus in
which it is inserted is activated; and
[0311] (ii) a sequence encoding a marker protein that is disrupted
by a nucleotide sequence with flanking recombinase sites that is
excised by the recombinase to generate an undisrupted marker gene.
This sequence can be inserted into a genetic locus that is
constitutively active, or into the same locus as the recombinase
DNA.
[0312] When the genetic locus in which the recombinase DNA is
inserted is activated, recombinase is synthesized and catalyzes
excision of the disrupting sequence from the marker DNA sequence,
permitting detection of the marker in the differentiated cell. When
transcription of the marker DNA is under control of a
constitutively active promoter, the marker can be detected even
when the locus in which the recombinase DNA is inserted is a
transiently activated locus that subsequently becomes deactivated.
(See Zinyk et al., Curr. Biol. (1998) 8:665-668; Dymecki et al.,
Dev. Biol. (1998) 201:57-65, each incorporated by reference in its
entirety). For example, the recombinase systems such as that of the
.lambda. integrase family can be used to implement this method. The
cre-loxP and FLO-FRT systems allow the activation or inactivation
of target sequences that operate as permanent markers in the
genomes of cells having passed certain points in development. The
use of these systems in fate mapping cells in animal development is
well known in the art; however, the use of recombinase-mediated
cell fate marking for the in vitro screening of stem cell
differentiation has not been described. Current fate mapping
techniques utilize two components: 1) a recombinase animal that
expresses the recombinase (Cre or FLP) in a gene-specific manner,
and 2) the indicator animal that has a transgene activated in the
presence of the recombinase in a permanent fashion e.g. such that
.beta.-gal is expressed in this and all cells derived from such a
cell regardless of their differentiated state. This recombinase can
be introduced into ES cells in gene trap vectors as described
above, and the recombinant ES cells can be used to produce an
assortment of individual recombinase mice that can provide a random
assortment of gametes harboring many gene trapped recombinase
genes. These gametes (sperm or eggs) can then be used with the
complementary gamete from the indicator animal to produce embryos,
embryoid bodies, or stem cells that leave a permanent marker of
having passed a given point in the developmental tree. Such
lineage-tracing stem cells have particular utility when the gene of
interest is only transiently expressed and therefore difficult to
detect. Libraries of stem cells in which such recombinase-based
markers are randomly inserted may be made and screened to identify
cell type associated gene trap markers. Alternatively, libraries of
stem cells in which such recombinase-based markers are targeted to
specific loci are useful in the screening assay of the present
invention for determining the conditions under which stem cells are
induced to express cell type-associated genes and differentiate
into a particular cell type.
* * * * *