Transcriptional profiling of stem cells and their multilineage differentiation

• US 9,062,346 B2
• Filed: 12/15/2010
• Issued: 06/23/2015
• Est. Priority Date: 04/01/2005
• Status: Active Grant

• Current Assignee: Wake Forest University Health Sciences
• Original Assignee: Wake Forest University Health Sciences
• Inventors: Hipp, Jason, Soker, Shay
• Primary Examiner(s): Li, Janice
• Application Number:
  US12/969,070
• Publication Number:
  US 20110081654A1
• Time in Patent Office: 1,651 Days
• Field of Search: None
• US Class Current: 1/1
• 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 human stem cell for differentiation into an osteogenic specific cell line, comprising:

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

(b) subjecting said human stem cell to differentiating conditions; and

then

(c) detecting in said stem cell differential expression of at least four osteogenic specific genes 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),

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

2. The method of claim 1, wherein said detecting of at least four osteogenic specific genes comprises detecting upregulation of expression of at least two genes selected from the group consisting of:

ICAM1, PPARG, SPARC, and BMP1,

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

3. The method of claim 2, wherein said detecting step comprises detecting upregulation of expression of at least two additional osteogenic specific genes selected from the group consisting of:

OMD, TIMP4, SOX4, CRYAB, SPP1, FOS, ITGAV, PRL, ITGA4, and SAS,

wherein upregulation of expression of said osteogenic specific genes indicates differentiation of said human stem cell into an osteogenic specific cell line.

4. The method of claim 1, wherein said human stem cell is a human amniotic fluid stem cell.

5. The method of claim 4, wherein said amniotic fluid stem cell does not form a teratoma when grown in vivo.

6. The method of claim 4, wherein said amniotic fluid stem cell does not form a teratoma within one month after intraarterial injection into a 6-8 week old mouse at a dose of 5×

10⁶ cells per mouse.

7. The method of claim 4, wherein said amniotic fluid stem cell is isolated from amniotic fluid between 14 and 18 weeks of gestation.

8. The method of claim 1, wherein said human stem cell is a human adipose-derived stem cell.

9. A method of screening a stem cell for differentiation into an osteogenic specific cell line, comprising:

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

(b) subjecting said stem cell to differentiating conditions; and

then

(c) detecting in said stem cell upregulation of expression of at least three osteogenic specific genes selected from the group consisting of;

ICAM1, PPARG, SPARC, and BMP1,

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

10. The method of claim 9, wherein said detecting of at least three osteogenic specific genes comprises detecting upregulation of expression of SPARC and BMP1,

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

11. The method of claim 10, wherein said detecting of at least three osteogenic specific genes further comprises detecting upregulation of expression of ICAM1 and PPARG,

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

12. The method of claim 11, wherein said stem cell is a human adipose-derived stem cell.

13. The method of claim 11, wherein said stem cell is a human stem cell.

14. The method of claim 13, wherein said human stem cell is a human amniotic fluid stem cell.

15. The method of claim 14, wherein said amniotic fluid stem cell does not form a teratoma when grown in vivo.

16. The method of claim 14, wherein said amniotic fluid stem cell does not form a teratoma within one month after intraarterial injection into a 6-8 week old mouse at a dose of 5×

10⁶ cells per mouse.

17. The method of claim 14, wherein said amniotic fluid stem cell is isolated from amniotic fluid between 14 and 18 weeks of gestation.