Transformed human pluripotent stem cells and associated methods

Patent metadata

Field	Value
Publication number	US-2016264935-A1
Application number	US-201615164528-A
Country	US
Kind code	A1
Filing date	May 25, 2016
Priority date	Sep 1, 2009
Publication date	Sep 15, 2016
Grant date	—

Abstract

The present disclosure provides transformed human pluripotent stem cell (t-hPSC). t-hPSCs are not dependent on Oct4 for renewal and survival, however exhibit a sensitivity to reduced levels of the transcription factor Nanog. Also provided are methods of culturing cells for use in a cell-based screening assay comprising placing one or more transformed human pluripotent stem cells into a receptacle and culturing said stem cells in the receptacle to form a monolayer of stem cells without cell overlap. Methods of screening compounds using t-hPSCs are also described.

First claim

1 . A transformed human pluripotent stem cell (t-hPSC) wherein the cell does not require bFGF for maintenance of an undifferentiated state.

2 . The transformed human pluripotent stem cell of claim 1 , wherein:
a) the cell co-expresses FGFR1 and IGFR1, b) the cell maintains expression of SSEA3 in the absence of bFGF, or c) the cell requires Nanog for self-renewal and cell survival.

3 . The transformed human pluripotent stem cell of claim 1 , wherein the cell exhibits reduced neuronal differentiation or reduced hematopoietic potential in vitro compared to a normal human pluripotent stem cell.

4 . The transformed human pluripotent stem cell of claim 1 , wherein t-hPSC-derived neural precursors do not form metastasis in vivo.

5 . The transformed human pluripotent stem cell of claim 1 , wherein the cell can be passaged as a single cell.

6 . A method of screening a compound for a biological activity comprising:
a) contacting one or more transformed human pluripotent stem cells (t-hPSCs) of claim 1 with the compound; b) detecting an effect of the compound on the one or more t-hPSCs, wherein the effect is indicative of the biological activity of the compound.

7 . The method of claim 6 , wherein the biological activity is cell differentiation activity or loss of pluripotency.

8 . The method of claim 7 , wherein step b) comprises detecting the emergence of progenitor or precursor cells from the one or more t-hPSCs that are otherwise refractory to differentiation.

9 . The method of claim 7 , wherein step b) comprises detecting the expression of one of more biomarkers selected from the group consisting of Oct4, Sox2, Nanog, SSEA3, SSEA4, TRA-1-60, TRA-1-81, IGF1 receptor, connexin 43, E-cadherin, Alkaline phosphatase, REX1, CRIPTO, CD24, CD90, CD29, CD9 and CD49f.

10 . The method of claim 6 , wherein the biological activity is anticancer activity and step b) comprises detecting a reduction in colony initiating cells.

11 . The method of claim 6 , wherein the biological activity is anticancer activity and step b) comprises detecting an increase in cell differentiation, an increase in loss of pluripotency, a decrease in cell proliferation, a decrease in cell number, a decrease in DNA content, a decrease in cell growth, an increase in cytotoxicity, or an increase in apoptosis.

12 . The method of claim 6 , further comprising comparing the effect of the compound on the one or more t-hPSCs determined in step b) to an effect of the compound on one or more normal stem cells (SCs).

13 . The method of claim 12 , wherein the effect of the compound on one or more normal stem cells (SCs) is determined by
c) contacting one or more stem cells (SCs) with the compound; d) detecting an effect of the compound on the one or more SCs, wherein the effect is indicative of the biological activity of the compound.

14 . The method of claim 13 , further comprising identifying compounds that exhibit a different level of biological activity on the one or more t-hPSCs compared to the one or more SCs.

15 . The method of claim 6 , further comprising placing one or more t-hPSCs into a receptacle and culturing the cells in the receptacle to form a monolayer prior to contacting the cells with the compound.

16 . The method of claim 15 , wherein a single t-hPSC is placed into the receptacle and the monolayer of t-hPSCs is a homogeneous clonal colony of the single t-hPSC.

17 . A cell culture comprising a plurality of transformed human pluripotent stem cells (t-hPSCs) of claim 1 .

18 . A method of identifying transformed pluripotent stem cells or transformed induced pluripotent stem cells comprising identifying stem cells that maintain expression of SSEA-3 when deprived of bFGF.

19 . The method of claim 18 comprising:
a) culturing pluripotent stem cells with bFGF;
b) analyzing the cells for SSEA-3 to provide a first level of SSEA-3 expression;
c) depleting the cell culture of bFGF;
d) analyzing the cells for SSEA-3 to provide at least a second level of SSEA-3 expression;
e) identifying those cells that maintain expression of SSEA-3 upon depletion of bFGF by comparing the first and second levels of SSEA-3 expression.

20 . A method of identifying transformed pluripotent stem cells comprising
a) inhibiting Oct4 expression in a population of pluripotent stem cells; and b) identifying undifferentiated cells that maintain expression of SSEA3.

Assignees

• Univ Mcmaster

Inventors

• Bhatia Mickie
• Werbowetski-Ogilvie Tamra
• Sachlos Eleftherios
• Fischer Russell Daniela
• Laronde Sarah
• Lee Jungbok
• Szabo Eva
• Risueno Ruth

Classifications

• G01N2500/10

  involving cells · CPC title

• C12Q1/04

  Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor {(C12Q1/6897 takes precedence)} · CPC title

• C12N5/0606 Primary

  Pluripotent embryonic cells, e.g. embryonic stem cells [ES] (embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696) · CPC title

• C12N2501/115

  Basic fibroblast growth factor (bFGF, FGF-2) · CPC title

• C12N5/0695

  Stem cells; Progenitor cells; Precursor cells · CPC title