Early developmental genomic assay for characterizing pluripotent stem cell utility and safety

Patent metadata

Field	Value
Publication number	US-10626445-B2
Application number	US-201815983209-A
Country	US
Kind code	B2
Filing date	May 18, 2018
Priority date	Jun 10, 2013
Publication date	Apr 21, 2020
Grant date	Apr 21, 2020

Abstract

The present invention generally relates to a set of early developmental reference data or “lineage scorecard” for stem cells, and methods, systems and kits to generate a lineage scorecard for predicting the functionality and suitability of stem cell lines. In some aspects, methods for generating a scorecard comprises measuring the gene expression of a plurality of early developmental genes, such as pluripotent, early ectoderm, early mesoderm and early endoderm genes to predict the pluripotency and differentiation potential of the stem cell line and its functionality and/or suitability for a desired use. In some embodiments, a reference scorecard can be compared with the test stem cell line scorecard to accurately predict the utility and/or identify specific characteristics of the stem cell line, e.g., to determine its suitability for downstream applications, e.g., therapeutic use, drug screening, toxicity assays, differentiation into a desired cell lineage, etc.

First claim

The invention claimed is:

1. An array composition for characterizing the differentiation potential of a pluripotent stem cell, wherein the array comprises a solid support and located on the solid support at assigned positions defined by x and y coordinates are at least 30 pairs of amplification primers and at least 30 oligonucleotides probes, and wherein the array comprises no more than 100 pairs of amplification primers and no more than 100 oligonucleotide probes,
wherein the oligonucleotide probes comprise an attached fluorescent dye and specifically hybridize to mRNA or cDNA of at least 30 early developmental genes selected from the group consisting of SEQ ID NO: 11, 14, 15, 19, 20, 21, 22, 23, 30, 32, 33, 34, 36, 37, 44, 45, 46, 48, 49, 53, 62, 63, 65, 66, 68, 70, 78, 84, 86, 87 and 88, and
wherein the at least 30 pairs of amplification primers that specifically amplify mRNA of a set of at least 30 early developmental genes selected from the group consisting of SEQ ID NO: 11, 14, 15, 19, 20, 21, 22, 23, 30, 32, 33, 34, 36, 37, 44, 45, 46, 48, 49, 53, 62, 63, 65, 66, 68, 70, 78, 84, 86, 87 and 88.

2. A method of determining the differentiation potential of a pluripotent stem cell line comprising;
providing the array of claim 1 ;
amplifying mRNA to produce cDNA, of said set of at least 30 early developmental genes selected from the group consisting of SEQ ID NO: 11, 14, 15, 19, 20, 21, 22, 23, 30, 32, 33, 34, 36, 37, 44, 45, 46, 48, 49, 53, 62, 63, 65, 66, 68, 70, 78, 84, 86, 87 and 88 in said pluripotent stem cell line, wherein the amount of cDNA produced corresponds to the amount of mRNA for each early developmental gene amplified;
measuring the level of cDNA for each of the at least 30 early developmental genes amplified in the pluripotent stem cell; and
comparing the level of cDNA expression of the measured set of at least 30 early developmental genes to the level of expression of the same early developmental genes in a control pluripotent stem cell sample, and based on this comparison, determining the differentiation potential of the pluripotent stem cell line.

3. A kit comprising:
a. the array of claim 1 ; and
b. reagents to carry out amplification of the mRNA of the at least 30 early developmental genes.

4. The array of claim 1 , wherein at least 4 of the 30 oligonucleotides probes or at least 30 pairs of amplification primers are ectoderm genes, at least 4 of the 30 oligonucleotides probes or at least 30 pairs of amplification primers are endoderm genes and at least 4 of the 30 oligonucleotides probes or at least 30 pairs of amplification primers are mesoderm genes.

5. A method of determining the differentiation potential of a pluripotent stem cell line comprising;
providing the array of claim 1 ; and
amplifying the mRNA, to produce cDNA, of a set of at least 12 early developmental genes selected from the group consisting of:
at least 4 ectoderm genes selected from the group consisting of SEQ ID NO: 11, 14, 15, 19, 20, 21 and 22;
at least 4 endoderm genes selected from the group consisting of SEQ ID NO: 23, 30, 32, 33, 34, 36, 37, 44, 45, 46, 48 and 49; and
at least 4 mesoderm genes selected from the group consisting of SEQ ID NO: 53, 62, 63, 65, 66, 68 and 70; in said pluripotent stem cell line, wherein the amount of cDNA produced corresponds to the amount of mRNA for each early developmental gene amplified;
measuring the level of cDNA for each of the at least 12 early developmental genes amplified in the pluripotent stem cell;
comparing the level of cDNA expression of the measured set of at least 12 early developmental genes to the level of expression of the same early developmental genes in a control pluripotent stem cell sample, and based on this comparison, determining the differentiation potential of the pluripotent stem cell line.

6. An array composition for characterizing the differentiation potential of a pluripotent stem cell, wherein the array comprises a solid support and located on the solid support at assigned positions defined by x and y coordinates are at least 12 pairs of amplification primers and at least 12 oligonucleotides, and wherein the array comprises no more than 100 pairs of amplification primers and no more than 100 oligonucleotide probes,
wherein the oligonucleotide probes have an attached fluorescent dye and hybridize to mRNA or cDNA of at least 12 early developmental genes, and wherein the at least 12 pairs of amplification primers amplify the mRNA of a set of at least 12 early developmental genes selected from the group of
at least 4 ectoderm genes selected from the group consisting of SEQ ID NO: 11, 14, 15, 19, 20, 21 and 22;
at least 4 endoderm genes selected from the group consisting of SEC ID NO: 23, 30, 32, 33, 34, 36, 37, 44, 45, 46, 48 and 49; and
at least 4 mesoderm genes selected from the group consisting of SEQ ID NO: SEQ ID NO: 53, 62, 63, 65, 66, 68 and 70.

7. A kit comprising:
a. the array of claim 6 ; and
b. reagents to carry out amplification of the mRNA of the at least 12 early developmental genes.

Assignees

• Harvard College

Inventors

• Meissner Alexander
• Tsankov Alexander
• Akopian Veronika

Classifications

• C12Q1/6876

  Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes · CPC title

• C12Q2600/158

  Expression markers · CPC title

• C12N2506/00

  Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q2600/16

  Primer sets for multiplex assays · CPC title