# US 9,435,786 B2 · Method for determining differentiation level of pluripotent stem cells

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 9,435,786 B2
- Document ID: US9435786B2
- Status: Granted
- Kind: B2
- Application number: 14/023,735
- Filed: 2013-09-11
- Published: 2014-03-13
- Granted: 2016-09-06
- Earliest claimed: 2012-09-13
- Assignees: Hamamatsu Photonics K.K.
- Applicants: Hamamatsu Photonics K.K.
- Inventors: Toyohiko Yamauchi; Norikazu Sugiyama; Tadashi Fukami; Hidenao Iwai
- Web page: https://patentgrep.com/patent/US9435786B2

## CPC

- G01N 33/4833: {of solid biological material, e.g. tissue samples, cell cultures (tissue in vivo A61B5/00; cell suspensions G01N33/48735)}
- C12N 5/0696: {Artificially induced pluripotent stem cells, e.g. iPS}
- C12Q 1/04: 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)}
- G01N 15/1456: {without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals}
- G06T 7/0012: {Biomedical image inspection}
- G06T 7/50: Depth or shape recovery
- G01N 2015/1006: {for cytology}
- G01N 2015/1497: {Particle shape}
- G06T 2207/10056: Microscopic image
- G06T 2207/30004: Biomedical image processing
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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
- G06T: IMAGE DATA PROCESSING OR GENERATION, IN GENERAL

## Abstract

The present invention provides a method for determining differentiation level of pluripotent stem cell, comprising a step of determining a flatness of cultured pluripotent stem cell, wherein the flatness is an indication.

## Claims

### Claim 1

A method comprising the steps of:
i) measuring flatness of at least one cultured pluripotent stem cell to determine whether the cultured pluripotent stem cell or a cultured pluripotent stem cell population is differentiated by applying a Student's t-test to compare the flatness of the cultured pluripotent stem cell or the cultured pluripotent stem cell population with those of undifferentiated pluripotent stem cells and differentiated cells, and
ii) identifying and removing at least one cultured pluripotent stem cell that is determined to be differentiated,
wherein the flatness of the cultured pluripotent stem cell or the cultured pluripotent stem cell population is measured from at least one tomographic image of a three-dimensional image of the cultured pluripotent stem cell or the cultured pluripotent stem cell population,
wherein the measured flatness is flatness of a surface of the cultured pluripotent stem cell roughly parallel to a bottom surface of the cultured pluripotent stem cell attached to a culture vessel or flatness of a surface of the cultured pluripotent stem cell population roughly parallel to a bottom surface of the cultured pluripotent stem cell population attached to a culture vessel, and
wherein the measured flatness of a differentiated cell or a differentiated cell population is statistically different from the measured flatness of an undifferentiated pluripotent stem cell or an undifferentiated pluripotent stem cell population.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the flatness of the cultured pluripotent stem cell or the cultured pluripotent stem cell population is measured from a single cultured pluripotent stem cell.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the flatness of the cultured pluripotent stem cell or the cultured pluripotent stem cell population is determined by calculating a standard deviation of height of the surface of one cultured pluripotent stem cell or the surface of the cultured pluripotent stem cell population,
wherein the height of the surface of one cultured pluripotent stem cell is a distance from a bottom surface of the cultured pluripotent stem cell attached to a culture vessel to the surface of the cultured pluripotent stem cell not attached to the culture vessel and roughly parallel to the bottom surface, and
wherein the height of the surface of the cultured pluripotent stem cell population is a distance from a bottom surface of the cultured pluripotent stem cell population attached to a culture vessel to the surface of the cultured pluripotent stem cell population not attached to the culture vessel and roughly parallel to the bottom surface.

### Claim 4 (depends on 3)

The method according to claim 3, wherein the calculated value for the flatness is standardized with a mean value of height of the surface of one cultured pluripotent stem cell or the surface of the cultured pluripotent stem cell population.

### Claim 5 (depends on 1)

The method according to claim 1, wherein the flatness of the cultured pluripotent stem cell or the cultured pluripotent stem cell population is determined by calculating a mean square height difference (MSHD) represented by formula (1):
MSHD(Δr)=<(z(x1,y1)−z(x2,y2))2> (I)
wherein z (x1, y1) is a height of a surface of the cultured pluripotent stem cell or the cultured pluripotent stem cell population at a first point, z (x2, y2) is a height of a surface of the cultured pluripotent stem cell or the cultured pluripotent stem cell population at a second point, and < > is an ensemble average, wherein Δr is distance in a horizontal direction between the first and the second point, and wherein (x1, y1) and (x2, y2) are in any combination that satisfies formula (II):
Δr=((x1−x2)2+(y1−y2)2)1/2 (II).

### Claim 6 (depends on 1)

The method according to claim 1, wherein the flatness of the cultured pluripotent stem cell or the cultured pluripotent stem cell population is determined using a reflection quantitative phase microscope.

### Claim 7 (depends on 1)

The method according to claim 1, wherein the flatness of the cultured pluripotent stem cell or the cultured pluripotent stem cell population is determined in a microscopic visual field unit.

### Claim 8 (depends on 7)

The method according to claim 7, wherein the flatness of the cultured pluripotent stem cell or the cultured pluripotent stem cell population is determined by calculating a standard deviation of height of the surface of one cultured pluripotent stem cell or the surface of the cultured pluripotent stem cell population,
wherein the height of the surface of one cultured pluripotent stem cell is a distance from a bottom surface of the cultured pluripotent stem cell attached to a culture vessel to the surface of the cultured pluripotent stem cell not attached to the culture vessel and roughly parallel to the bottom surface, and
wherein the height of the surface of the cultured pluripotent stem cell population is a distance from a bottom surface of the cultured pluripotent stem cell population attached to a culture vessel to the surface of the cultured pluripotent stem cell population not attached to the culture vessel and roughly parallel to the bottom surface.

### Claim 9 (depends on 7)

The method according to claim 7, wherein the flatness of the cultured pluripotent stem cell or the cultured pluripotent stem cell population is determined by calculating a mean square height difference (MSHD) represented by formula (1):
MSHD(Δr)=<(z(x1,y1)−z(x2,y2))2> (I)
wherein z (x1, y1) is a height of a surface of the cultured pluripotent stem cell or the cultured pluripotent stem cell population at a first point, z (x2, y2) is a height of a surface of the cultured pluripotent stem cell or the cultured pluripotent stem cell population at a second point, and < > is an ensemble average, wherein Δr is distance in a horizontal direction between the first and the second point, and wherein (x1, y1) and (x2, y2) are in any combination that satisfies formula (II):
Δr=((x1−x2)2+(y1−y2)2)1/2 (II).

### Claim 10 (depends on 7)

The method according to claim 7, wherein the calculated value for the flatness is standardized with a mean value of height of the surface of one cultured pluripotent stem cell or the surface of the cultured pluripotent stem cell population.

### Claim 11 (depends on 7)

The method according to claim 7, wherein the flatness of the cultured pluripotent stem cell or the cultured pluripotent stem cell population is determined using a reflection quantitative phase microscope.

### Claim 12 (depends on 1)

The method according to claim 1, wherein the flatness of the cultured pluripotent stem cell or the cultured pluripotent stem cell population is determined in a region of interest unit within a microscopic visual field.

### Claim 13 (depends on 12)

The method according to claim 12, wherein the flatness of the cultured pluripotent stem cell or the cultured pluripotent stem cell population is determined by calculating a standard deviation of height of the surface of one cultured pluripotent stem cell or the surface of the cultured pluripotent stem cell population,
wherein the height of the surface of one cultured pluripotent stem cell is a distance from a bottom surface of the cultured pluripotent stem cell attached to a culture vessel to the surface of the cultured pluripotent stem cell not attached to the culture vessel and roughly parallel to the bottom surface, and
wherein the height of the surface of the cultured pluripotent stem cell population is a distance from a bottom surface of the cultured pluripotent stem cell population attached to a culture vessel to the surface of the cultured pluripotent stem cell population not attached to the culture vessel and roughly parallel to the bottom surface.

### Claim 14 (depends on 12)

The method according to claim 12, wherein the flatness of the cultured pluripotent stem cell or the cultured pluripotent stem cell population is determined by calculating a mean square height difference (MSHD) represented by formula (1):
MSHD(Δr)=<(z(x1,y1)−z(x2,y2))2> (I)
wherein z (x1, y1) is a height of a surface of the cultured pluripotent stem cell or the cultured pluripotent stem cell population at a first point, z (x2, y2) is a height of a surface of the cultured pluripotent stem cell or the cultured pluripotent stem cell population at a second point, and < > is an ensemble average, wherein Δr is distance in a horizontal direction between the first and the second point, and wherein (x1, y1) and (x2, y2) are in any combination that satisfies formula (II):
Δr=((x1−x2)2+(y1−y2)2)1/2 (II).

### Claim 15 (depends on 12)

The method according to claim 12, wherein the calculated value for the flatness is standardized with a mean value of height of the surface of one cultured pluripotent stem cell or the surface of the cultured pluripotent stem cell population.

### Claim 16 (depends on 12)

The method according to claim 12, wherein the flatness of the cultured pluripotent stem cell or the cultured pluripotent stem cell population is determined using a reflection quantitative phase microscope.