Transcriptional profiling of stem cells and their multilineage differentiation

• US 20060246488A1
• Filed: 03/31/2006
• Published: 11/02/2006
• Est. Priority Date: 04/01/2005
• Status: Active Grant

• Current Assignee: Wake Forest University Health Sciences
• Original Assignee: Wake Forest University Health Sciences
• Inventors: Soker, Shay, Hipp, Jason
• Granted Patent: US 7,883,847 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

Abstract

The present invention concerns methods of screening cells for differentiation or de-differentiation, and/or for status as a pluripotent or multipotent (e.g., “stem”) cell, by detecting the differential expression (e.g., upregulation, downregulation) of genes.

Claims

1. A method of screening a pluripotent or multipotent cell for differentiation into a (i) heptogenic, (ii) myogenic, (iii) osteogenic, or (iv) endothelial specific cell line, comprising:

(a) providing a cell for which differentiation is to be determined, then

(b) subjecting said cell to differentiating conditions; and

then

(c) detecting in said cell differential expression of;

(i) at least one hepatogenic specific gene selected from the group consisting of stearlyl-CoA desaturase (SCD), 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMGCR), insulin inducd gene 1 (INSIG1), chromosome 20 open reading frame 97 (C20orf97), lipase A (LIPA), fatty acid desaturase 1 (FADS1), 7-dehydrocholesterol reductase (DHCR7), apolipoprotein D (APOD), squalene epoxidase (SQLE), cholesterol 25-hydroxylase (CH25H), lipin 1 (LPIN1), insulin induced gene 1 (INSIG1), flavin cntaining monooxygenase 1 (FMO1), aldo-keto reductase family 1 member 1C (AKR1C1), insulin-like growth factor 2 receptor (IGFR2R), ATP-binding cassette sub-family A member 1 (ABCA1), X-box binding protein 1 (XBP1) and mucin 1 (MUC1);

(ii) at least one myogenic specific gene selected from the group consisting of insulin-like grwth factor binding protein 3 (IGFBP3), caldesmonin 1 (CALD1), a disintegrin and metallproteinase domain 12 (ADAM12), transglutaminase 2 (TGM2), tumor necrosis factor receptor superfamily member 11b (TNFRSF11B), protein kinase H11 (H11), cardiac muscle alpha actin (ACTC), and sarcoglycan delta (SGCD);

at least one osteogenic specific gene selected from the group consisting of;

intracellular adhesion molecule 1 (ICAM1), osteomodulin (OMD), tissue inhibitor of metalloporteinase 4 (TIMP4), sex determining region Y box 4 (SOX4), secreted phosphoprotein 1 (SPP1), v-fos FBJ murine osteosarcoma viral oncogene homolog (FOS), alpha V integrin (ITGAV), prolactin (PRL), alpha 4 integrin (ITGA4), peroxisome proliferative activated receptor gamma (PPARG), secreted protein acidic cystein-rich (SPARC) sarcoma amplified sequence (SAS), and bone morphogenetic protein 1 (BMP1),

(iii) at least one osteogenic specific gene selected from the group consisting of;

intracellular adhesion molecule 1 (ICAM1), osteomodulin (OMD), tissue inhibitor of metalloproteinase 4 (TIMP4), sex determining region Y box 4 (SOX4), crystalin alpha B (CRYAB), secreted phosphoprotein 1 (SPP1), v-fos FBJ murine steosarcoma viral oncogene homolog (FOS), alpha V integrin (ITGAV), prolactin (PRL), alpha 4 integrin (ITGA4), peroxisome proliferative activated receptor gamma (PPARG), secreted protein, acidic, cystein-rich (SPARC), sarcoma amplified sequence (SAS), and bone morphogenetic protein 1 (BMP1), or

(iv) at least one endothelial specific gene selected from the group consisting of pentaxin-related gene rapidly induced by IL-1 beta (PTX3), selenprotein P plasma 1 (SEPP1), tissue factor pathway inhibitor (TFP1), angiopietin 1 (ANGPT1), angiopoietin-like 2 (ANGPTL2), 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMGCR), kruppel-like factor 4 (KLF4), endothelial differentiation lysophosphatidic acid G-protein coupled receptor 2 (EDG2), matrix metalloporiteinase 14 (MPP14), neronal cell adhesion molecule (NRCAM), interleukin 6 (IL6), and tumor necrosis factor, alpha-induced protein 6 (TNFAIP6);

wherein (i) upregulation of expression of said at least one hepatogenic specific gene indicates differentiation of said cell into a heptogenic specific cell line, (ii) upregulation of expression of said at least one myogenic specific gene indicates differentiation of said cell into a myogenic specific cell line, (iii) upregulation of expression of said at least one osteogenic specific gene indicates differentiation of said cell into an osteogenic specific cell line, or (iv) upregulation of said expression of said at least one endothelial specific gene indicates differentiation of said cell into and endothelial specific cell line.

2. The method of claim 1, wherein said detecting step comprises detecting upregulation of expression of (i) at least one hepatogenic specific gene selected from the group consisting of SCD, HMGCR, INSIG1, C20orf97, LIPA, FADS1, DHCR7, APOD, SQLE, CH25H, LPIN1, INSIG1, FMO1, AKR1C1, IGFR2R, ABCA1, XBP1 and MUC1;

wherein upregulation of said at least one hepatogenic specific gene indicates differentiation of said cell into a heptogenic specific cell line.

3. The method of claim 1, wherein said detecting step comprises detecting upregulation of expression of (i) at least two hepatogenic specific genes selected from the group consisting of SCD, HMGCR, INSIG1, C20orf97, LIPA, FADS1, DHCR7, APOD, SQLE, CH25H, LPIN1, INSIG1, FMO1, AKR1C1, IGFR2R, ABCA1, XBP1 and MUC1;

wherein upregulation of said at least two hepatogenic specific genes indicates differentiation of said cell into a heptogenic specific cell line.

4. The method of claim 1, wherein said detecting step comprises detecting upregulation of expression of (ii) at least one myogenic specific gene selected from the group consisting of IGFBP3, CALD1, ADAM12, TGM2, TNFRSF11B, H11, ACTC, and SGCD;

wherein upregulation of expression of said at least one myogenic specific gene indicates differentiation of said cell into a myogenic specific cell line

5. The method of claim 1, wherein said detecting step comprises detecting upregulation of expression of (ii) at least two myogenic specific genes selected from the group consisting of IGFBP3, CALD1, ADAM12, TGM2, TNFRSF11B, H11, ACTC, and SGCD;

wherein upregulation of expression of said at least two myogenic specific gene indicates differentiation of said cell into a myogenic specific cell line.

6. The method of claim 1, wherein said detecting step comprises detecting upregulation of expression of (iii) at least one osteogenic specific gene selected from the group consisting of:

ICAM1, OMD, TIMP4, SOX4, CRYAB, SPP1, FOS, ITGAV, PRL, ITGA4, PPARG, SPARC, SAS, and BMP1,

wherein upregulation of expression of said at least one osteogenic specific gene indicates differentiation of said cell into an osteogenic specific cell line.

7. The method of claim 1, wherein said detecting step comprises detecting upregulation of expression of (iii) at least two osteogenic specific genes selected from the group consisting of:

ICAM1, OMD, TIMP4, SOX4, CRYAB, SPP1, FOS, ITGAV, PRL, ITGA4, PPARG, SPARC, SAS, and BMP1,

wherein upregulation of expression of said at least two osteogenic specific genes indicates differentiation of said cell into an osteogenic specific cell line.

8. The method of claim 1, wherein said detecting step comprises detecting upregulation of expression of (iv) at least one endothelial specific gene selected from the group consisting of PTX3, SEPP1, TFPI, ANGPT1, ANGPTL2, HMGCR, KLF4, EDG2, MPP14, NRCAM, IL6, and TNFAIP6;

wherein upregulation of said expression of said at least one endothelial specific gene indicates differentiation of said cell into and endothelial specific cell line.

9. The method of claim 1, wherein said detecting step comprises detecting upregulation of expression of (iv) at least two endothelial specific genes selected from the group consisting of PTX3, SEPP1, TFP1, ANGPT1, ANGPTL2, HMGCR, KLF4, EDG2, MPP14, NRCAM, IL6, and TNFAIP6;

wherein upregulation of said expression of said at least two endothelial specific genes indicates differentiation of said cell into and endothelial specific cell line.

10. The methd of claim 1, wherein said cell is a stem cell.

14. The method of claim 1, wherein said detecting step comprises detecting down regulation of expression of (ii) at least one myogenic specific gene selected from the group consisting of IGFBP3, CALD1, ADAM12, TGM2, TNFRSF11B, H11, ACTC, and SGCD;

wherein down regulation of expression of said at least one myogenic specific gene indicates de-differentiation of said myogenic specific cell line.

20. The methd of claim 1, wherein said cell de-differentiates to a pluripotent or multipotent stem cell.

11. A method of screening a (i) heptogenic, (ii) myogenic, (iii) osteogenic, or (iv) endothelial specific cell for de-differentiation into a pluripotent or multipotent cell, comprising:

(a) providing a pluripotent or multipotent cell for which de-differentiation is to be determined, then

(b) subjecting said cell to de-differentiating conditions; and

then

(c) detecting in said cell differential expression of;

(i) at least one hepatogenic specific gene selected from the group consisting of stearlyl-CoA desaturase (SCD), 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMGCR), insulin inducd gene 1 (INSIG1), chromosome 20 open reading frame 97 (C20orf97), lipase A (LIPA), fatty acid desaturase 1 (FADS1), 7-dehydrocholesterol reductase (DHCR7), apolipoprotein D (APOD), squalene epoxidase (SQLE), cholesterol 25-hydroxylase (CH25H), lipin 1 (LPIN1), insulin induced gene 1 (INSIG1), flavin cntaining monooxygenase 1 (FMO1), aldo-keto reductase family 1 member 1C (AKR1C1), insulin-like growth factor 2 receptor (IGFR2R), ATP-binding cassette sub-family A member 1 (ABCA1), X-box binding protein 1 (XBP1) and mucin 1 (MUC1);

(ii) at least one myogenic specific gene selected from the group consisting of insulin-like grwth factor binding protein 3 (IGFBP3), caldesmonin 1 (CALD1), a disintegrin and metallproteinase domain 12 (ADAM12), transglutaminase 2 (TGM2), tumor necrosis factor receptor superfamily member 11b (TNFRSF11B), protein kinase H11 (H11), cardiac muscle alpha actin (ACTC), and sarcoglycan delta (SGCD);

(iii) at least one osteogenic specific gene selected from the group consisting of;

intracellular adhesion molecule 1 (ICAM1), osteomodulin (OMD), tissue inhibitor of metalloporteinase 4 (TIMP4), sex determining region Y box 4 (SOX4), secreted phosphoprotein 1 (SPP1), v-fos FBJ murine osteosarcoma viral oncogene homolog (FOS), alpha V integrin (ITGAV), prolactin (PRL), alpha 4 integrin (ITGA4), peroxisome proliferative activated receptor gamma (PPARG), secreted protein acidic cystein-rich (SPARC) sarcoma amplified sequence (SAS), and bone morphogenetic protein 1 (BMP1), (iii) at least one osteogenic specific gene selected from the group consisting of intracellular adhesion molecule 1 (ICAM1), osteomodulin (OMD), tissue inhibitor of metalloproteinase 4 (TIMP4), sex determining region Y box 4 (SOX4), crystalin alpha B (CRYAB), secreted phosphoprotein 1 (SPP1), v-fos FBJ murine steosarcoma viral oncogene homolog (FOS), alpha V integrin (ITGAV), prolactin (PRL), alpha 4 integrin (ITGA4), peroxisome proliferative activated receptor gamma (PPARG), secreted protein, acidic, cystein-rich (SPARC), sarcoma amplified sequence (SAS), and bone morphogenetic protein 1 (BMP1), or

(iv) at least one endothelial specific gene selected from the group consisting of pentaxin-related gene rapidly induced by IL-1 beta (PTX3), selenprotein P plasma 1 (SEPP1), tissue factor pathway inhibitor (TFP1), angiopietin 1 (ANGPT1), angiopoietin-like 2 (ANGPTL2), 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMGCR), kruppel-like factor 4 (KLF4), endothelial differentiation lysophosphatidic acid G-protein coupled receptor 2 (EDG2), matrix metalloporiteinase 14 (MPP14), neronal cell adhesion molecule (NRCAM), interleukin 6 (IL6), and tumor necrosis factor, alpha-induced protein 6 (TNFAIP6);

wherein (i) down regulation of expression of said at least one hepatogenic specific gene indicates de-differentiation of a heptogenic specific cell line, (ii) downregulation of expression of said at least one myogenic specific gene indicates de-differentiation of said myogenic specific cell line, (iii) downregulation of expression of said at least one osteogenic specific gene indicates de-differentiation of said osteogenic specific cell line, or (iv) downregulation of expression of said at least one endothelial specific gene indicates de-differentiation of said endothelial specific cell line.

12. The method of claim 11, wherein said detecting step comprises detecting down regulation of expression of (i) at least one hepatogenic specific gene selected from the group consisting of SCD, HMGCR, INSIG1, C20orf97, LIPA, FADS1, DHCR7, APOD, SQLE, CH25H, LPIN1, INSIG1, FMO1, AKR1C1, IGFR2R, ABCA1, XBP1 and MUC1;

wherein down regulation of said at least one hepatogenic specific gene indicates de-differentiation of said a heptogenic specific cell line.

13. The method of claim 11, wherein said detecting step comprises detecting down regulation of expression of (i) at least two hepatogenic specific genes selected from the group consisting of SCD, HMGCR, INSIG1, C20orf97, LIPA, FADS1, DHCR7, APOD, SQLE, CH25H, LPIN1, INSIG1, FMO1, AKR1C1, IGFR2R, ABCA1, XBP1 and MUC1;

wherein down regulation of said at least two hepatogenic specific genes indicates de-differentiation of said heptogenic specific cell line.

15. The method of claim 11, wherein said detecting step comprises detecting down regulation of expression of (ii) at least two myogenic specific genes selected from the group consisting of IGFBP3, CALD1, ADAM12, TGM2, TNFRSF11B, H11, ACTC, and SGCD;

wherein down regulation of expression of said at least two myogenic specific gene indicates de-differentiation of said myogenic specific cell line.

16. The method of claim 11, wherein said detecting step comprises detecting down regulation of expression of (iii) at least one osteogenic specific gene selected from the group consisting of:

ICAM1, OMD, TIMP4, SOX4, CRYAB, SPP1, FOS, ITGAV, PRL, ITGA4, PPARG, SPARC, SAS, and BMP1,

wherein down regulation of expression of said at least one osteogenic specific gene indicates de-differentiation of said osteogenic specific cell line.

17. The method of claim 11, wherein said detecting step comprises detecting down regulation of expression of (iii) at least two osteogenic specific genes selected from the group consisting of:

ICAM1, OMD, TIMP4, SOX4, CRYAB, SPP1, FOS, ITGAV, PRL, ITGA4, PPARG, SPARC, SAS, and BMP1,

wherein down regulation of expression of said at least two osteogenic specific genes indicates de-differentiation of said an osteogenic specific cell line.

18. The method of claim 11, wherein said detecting step comprises detecting down regulation of expression of (iv) at least one endothelial specific gene selected from the group consisting of PTX3, SEPP1, TFP1, ANGPT1, ANGPTL2, HMGCR, KLF4, EDG2, MPP14, NRCAM, IL6, and TNFAIP6;

wherein down regulation of said expression of said at least one endothelial specific gene indicates de-differentiation of said endothelial specific cell line.

19. The method of claim 11, wherein said detecting step comprises detecting down regulation of expression of (iv) at least two endothelial specific genes selected from the group consisting of PTX3, SEPP1, TFP1, ANGPT1, ANGPTL2, HMGCR, KLF4, EDG2, MPP14, NRCAM, IL6, and TNFAIP6;

wherein down regulation of said expression of said at least two endothelial specific genes indicates de-differentiation of said endothelial specific cell line.

21. A combination comprising a plurality of cDNAs that are differentially expressed in a lineage specific cell line, wherein the plurality of cDNAs consist of cDNAs encoding:

(i) at least two hepatogenic specific genes selected from the group consisting of stearlyl-CoA desaturase (SCD), 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMGCR), insulin inducd gene 1 (INSIG1), chromosome 20 open reading frame 97 (C20orf97), lipase A (LIPA), fatty acid desaturase 1 (FADS1), 7-dehydrocholesterol reductase (DHCR7), apolipoprotein D (APOD), squalene epoxidase (SQLE), cholesterol 25-hydroxylase (CH25H), lipin 1 (LPIN1), insulin induced gene 1 (INSIG1), flavin cntaining monooxygenase 1 (FMO1), aldo-keto reductase family 1 member 1C (AKR1C1), insulin-like growth factor 2 receptor (IGFR2R), ATP-binding cassette sub-family A member 1 (ABCA1), X-box binding protein 1 (XBP1) and mucin 1 (MUC1), or the complements thereof;

(ii) at least two myogenic specific genes selected from the group consisting of insulin-like grwth factor binding protein 3 (IGFBP3), caldesmonin 1 (CALD1), a disintegrin and metallproteinase domain 12 (ADAM12), transglutaminase 2 (TGM2), tumor necrosis factor receptor superfamily member 11b (TNFRSF11B), protein kinase H11 (H11), cardiac muscle alpha actin (ACTC), and sarcoglycan delta (SGCD);

at least one osteogenic specific gene selected from the group consisting of;

intracellular adhesion molecule 1 (ICAM1), osteomodulin (OMD), tissue inhibitor of metalloporteinase 4 (TIMP4), sex determining region Y box 4 (SOX4), secreted phosphoprotein 1 (SPP1), v-fos FBJ murine osteosarcoma viral oncogene homolog (FOS), alpha V integrin (ITGAV), prolactin (PRL), alpha 4 integrin (ITGA4), peroxisome proliferative activated receptor gamma (PPARG), secreted protein acidic cystein-rich (SPARC) sarcoma amplified sequence (SAS), and bone morphogenetic protein 1 (BMP1), or the complements thereof,

(iii) at least two osteogenic specific genes selected from the group consisting of;

intracellular adhesion molecule 1 (ICAM1), osteomodulin (OMD), tissue inhibitor of metalloporteinase 4 (TIMP4), sex determining region Y box 4 (SOX4), secreted phosphoprotein 1 (SPP1), v-fos FBJ murine osteosarcoma viral oncogene homolog (FOS), alpha V integrin (ITGAV), prolactin (PRL), alpha 4 integrin (ITGA4), peroxisome proliferative activated receptor gamma (PPARG), secreted protein acidic cystein-rich (SPARC) sarcoma amplified sequence (SAS), and bone morphogenetic protein 1 (BMP1), (iii) at least one osteogenic specific gene selected from the group consisting of;

intracellular adhesion molecule 1 (ICAM1), osteomodulin (OMD), tissue inhibitor of metalloproteinase 4 (TIMP4), sex determining region Y box 4 (SOX4), crystalin alpha B (CRYAB), secreted phosphoprotein 1 (SPP1), v-fos FBJ murine steosarcoma viral oncogene homolog (FOS), alpha V integrin (ITGAV), prolactin (PRL), alpha 4 integrin (ITGA4), peroxisome proliferative activated receptor gamma (PPARG), secreted protein, acidic, cystein-rich (SPARC), sarcoma amplified sequence (SAS), and bone morphogenetic protein 1 (BMP1), or the complements thereof, or

(iv) at least two endothelial specific genes selected from the group consisting of pentaxin-related gene rapidly induced by IL-1 beta (PTX3), selenprotein P plasma 1 (SEPP1), tissue factor pathway inhibitor (TFPI), angiopietin 1 (ANGPT1), angiopoietin-like 2 (ANGPTL2), 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMGCR), kruppel-like factor 4 (KLF4), endothelial differentiation lysophosphatidic acid G-protein coupled receptor 2 (EDG2), matrix metalloporiteinase 14 (MPP14), neronal cell adhesion molecule (NRCAM), interleukin 6 (IL6), and tumor necrosis factor, alpha-induced protein 6 (TNFAIP6), or the complements thereof.

22. The combination of claim 21, wherein the cDNAs are immobilized on a substrate.