Methods and compositions for identifying undifferentiated stem cells and assessing cell health

Patent metadata

Field	Value
Publication number	US-9528154-B2
Application number	US-81248909-A
Country	US
Kind code	B2
Filing date	Jan 12, 2009
Priority date	Jan 10, 2008
Publication date	Dec 27, 2016
Grant date	Dec 27, 2016

Abstract

Disclosed herein are methods and compositions for the identification of teloRNA marks to assess the differentiation status of an individual stem cell or a population of stem cells, to diagnose whether and to what extent a stem cell or stem cell culture has already initiated cell differentiation, and to monitor the differentiation status of an individual stem cell or a stem cell culture during passage. The use of these methods and compositions to monitor the pluripotency and differentiation status of a stem cell or stem cell culture during differentiation enables the identification of undifferentiated and pluripotent stem cells prior to the initiation of differentiation. The methods and compositions can also be used to assess and maintain cell viability; to identify cells or a population of cells that are in a state of poor cell health; and to reduce cell growth or treat a diseased cell including, for example, pre-cancerous cells, cancerous cells, apoptotic cells, aging cells, cells undergoing stress, and otherwise diseased or dysfunctional cells.

First claim

What is claimed is:

1. A method for determining the differentiation state of a pluripotent stem cell, wherein said stem cell is one of a population of stem cells, said method comprising:
(a) contacting said stem cell with a heterogeneous Cot-1 nucleic acid probe under conditions for hybridization of the heterogeneous Cot-1 nucleic acid probe to a ribonucleic acid molecule in the nucleus of said stem cell; and
(b) detecting the hybridization of said heterogeneous Cot-1 nucleic acid probe to the ribonucleic acid molecule in the stem cell, wherein the hybridization of the heterogeneous Cot-1 nucleic acid probe to the ribonucleic acid molecule in the stem cell is detected as a teloRNA mark in situ using fluorescent in situ hybridization (FISH),
wherein the presence of two teloRNA marks, one on each sex chromosome, identifies said stem cell as a stem cell that is undifferentiated and the presence of only one teloRNA mark on an inactive X chromosome in a female stem cell or a Y chromosome in a male stem cell identifies said stem cell as a stem cell that is differentiated.

2. The method of claim 1 , wherein said Cot-1 nucleic acid probe hybridizes to a telomeric repeat sequence or a subtelomeric sequence.

3. The method of claim 2 , wherein said Cot-1 probe comprises at least one copy of the telomeric repeat sequence TAACCC, or a frameshift thereof.

4. The method of claim 1 , wherein said cell is an embryonic stem cell.

5. A method for determining the differentiation state of a pluripotent stem cell, wherein said stem cell is one of a population of stem cells, said method comprising:
(a) contacting said stem cell with a telomeric nucleic acid probe comprising at least one copy of a TAACCC nucleic acid sequence under conditions for hybridization of the telomeric nucleic acid probe to a ribonucleic acid molecule in the nucleus of said stem cell; and
(b) detecting the hybridization of said telomeric nucleic acid probe to the ribonucleic acid molecule in the stem cell in situ using fluorescent in situ hybridization (FISH), wherein the hybridization of the telomeric nucleic acid probe to the ribonucleic acid molecule in the stem cell is detected as a teloRNA mark, wherein the presence of two teloRNA marks, one on each sex chromosome, identifies said stem cell as a stem cell that is undifferentiated and the presence of only one teloRNA mark on an inactive X chromosome in a female stem cell or a Y chromosome in a male stem cell identifies said stem cell as a stem cell that is differentiated.

6. The method of claim 5 , wherein the hybridization is at high stringency.

7. The method of claim 5 , wherein the nucleus is undenatured.

8. The method of claim 5 , wherein said telomeric nucleic acid probe hybridizes to a telomeric repeat sequence.

9. The method of claim 5 , wherein said telomeric nucleic acid probe comprises a frameshift of said TAACCC nucleic acid sequence.

10. The method of claim 5 , wherein said telomeric nucleic acid probe comprises three to seven copies of the nucleic acid sequence TAACCC or a frameshift thereof.

11. The method of claim 5 , wherein said cell is an embryonic stem cell.

12. The method of claim 5 , further comprising determining the presence or absence of at least one X-chromosome inactivation (XCI) marker in said stem cell, wherein the presence of said XCI marker identifies said stem cell in said population of stem cells as a stem cell that is differentiated and the absence of said XCI marker identifies said stem cell as a cell that is undifferentiated.

13. The method of claim 5 , further comprising detecting at least one polypeptide pluripotency marker selected from the group consisted of Oct4, Nanog, Rex1, stage specific embryonic antigen (SSEA)-1, SSEA-3, and SSEA-4, wherein the presence of a polypeptide pluripotency marker identifies said stem cell as a stem cell that is undifferentiated.

14. A method for determining the differentiation state of a pluripotent stem cell culture, said method comprising:
(a) contacting said stem cell culture with a telomeric nucleic acid probe comprising at least one copy of a TAACCC nucleic acid sequence or a heterogeneous Cot-1 nucleic acid probe under conditions for hybridization of the telomeric nucleic acid probe or Cot-1 nucleic acid probe to ribonucleic acid molecules in the nuclei of stem cells in said stem cell culture; and
(b) detecting the hybridization of said telomeric nucleic acid probe or Cot-1 nucleic acid probe to the ribonucleic acid molecules in the stem cells in situ using fluorescent in situ hybridization (FISH) in said stem cell culture, wherein the hybridization of the telomeric nucleic acid probe or Cot-1 nucleic acid probe to the ribonucleic acid molecules in the stem cells appears as a teloRNA mark, wherein the presence of two teloRNA marks, one on each sex chromosome, in at least 5% of the cells in said stem cell culture identifies said stem cell culture as undifferentiated.

15. The method of claim 1 , wherein said cell is an induced pluripotent stem cell (iPS).

16. The method of claim 5 , wherein said cell is an induced pluripotent stem cell (iPS).

Assignees

• Lee Jeannie T
• Zhang Li-Feng
• Massachusetts Gen Hospital

Inventors

• Lee Jeannie T
• Zhang Li-Feng

Classifications

• C12Q2600/158

  Expression markers · CPC title

• C12Q1/6881 Primary

  for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes · CPC title