# US 10,501,811 B2 · Method for sorting tissue cells

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- Number: US 10,501,811 B2
- Document ID: US10501811B2
- Status: Granted
- Kind: B2
- Application number: 15/568,209
- Filed: 2016-04-22
- Published: 2018-04-12
- Granted: 2019-12-10
- Earliest claimed: 2015-04-22
- Assignees: Kyoto University
- Applicants: Kyoto University
- Inventors: Yoshinori Yoshida; Hirohide Saito; Kenji Miki; Kei Endo; Seiya Takahashi
- Web page: https://patentgrep.com/patent/US10501811B2

## CPC

- C12Q 1/6888: for detection or identification of organisms
- C12N 5/067: {Hepatocytes}
- C12N 5/0676: {Pancreatic cells}
- C12N 5/069: {Vascular Endothelial cells}
- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C12N 15/67: General methods for enhancing the expression
- C12Q 1/68: involving nucleic acids
- C12N 2506/45: from artificially induced pluripotent stem cells
- C12Q 2600/178: miRNA, siRNA or ncRNA
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

An object of the present invention is to provide a method for increasing the purity of a type of tissue cell such as an endothelial cell, a hepatocyte, or an insulin-producing cell. The present invention solves the problem by providing a method comprising a step of introducing, into a cell population, an mRNA comprising a nucleic acid sequence recognized by an miRNA specifically expressed in endothelial cells, hepatocytes, or insulin-producing cells.

## Claims

### Claim 1

A method for producing insulin-producing cells, comprising:
(a) producing, from a pluripotent stem cell, a cell population containing insulin-producing cells;
(b) introducing, into the cell population of the step (a), an miRNA-responsive mRNA consisting of sequences comprising (i) a nucleic acid recognized specifically by miR-375 and (ii) a nucleic acid corresponding to a coding region of a first marker gene, wherein the miRNA-responsive mRNA is introduced into the cell population in a form of a synthetic RNA molecule; and
(c) sorting a cell having a low level of translation of the first marker gene of the step (b).

### Claim 2 (depends on 1)

The method according to claim 1, wherein the miRNA-responsive mRNA comprises a Cap structure and a polyA tail.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the nucleic acid recognized specifically by miR-375 is included in a 5′ UTR or 3′ UTR of the coding region of the first marker gene in the miRNA-responsive mRNA.