# US 9,518,250 B2 · Method for the ex vivo expansion of hematopoietic stem and progenitor cells

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- Number: US 9,518,250 B2
- Document ID: US9518250B2
- Status: Granted
- Kind: B2
- Application number: 14/346,160
- Filed: 2012-09-21
- Published: 2014-09-25
- Granted: 2016-12-13
- Earliest claimed: 2011-09-22
- Assignees: Nohia Therapeutics Australia Pty Ltd
- Applicants: Cytomatrix Pty Ltd
- Inventors: Melinda L. Tursky; Mark. A. Kirkland
- Web page: https://patentgrep.com/patent/US9518250B2

## CPC

- C12N 5/0647: {Haematopoietic stem cells; Uncommitted or multipotent progenitors}
- A61P 7/00: Drugs for disorders of the blood or the extracellular fluid
- C12N 2501/125: Stem cell factor [SCF], c-kit ligand [KL]
- C12N 2501/145: Thrombopoietin [TPO]
- C12N 2501/22: Colony stimulating factors (G-CSF, GM-CSF)
- C12N 2501/2306: Interleukin-6 (IL-6)
- C12N 2501/26: Flt-3 ligand (CD135L, flk-2 ligand)
- C12N 2501/59: Lectins
- C12N 2501/998: Proteins not provided for elsewhere
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

The present invention relates to hematopoietic cells, and more specifically to methods for long-term in vitro culturing and ex vivo expansion of hematopoietic cells. The present invention also provides compositions useful for culturing cells, such as media for culturing hematopoietic cells, specifically haematopoietic stem cells (HSC) and haematopoietic progenitor cells (HPC). The present invention further provides compositions including growth factor combinations and methods utilizing altered growth and environmental conditions that are applicable in vitro culturing and to ex vivo expansion of HSC and/or HPC.

## Claims

### Claim 1

A method of extending ex vivo viability of a HPC and/or HSC population, or culturing or expanding a HPC and/or HSC population said method comprising culturing the HPC and/or HSC population in the presence of at least one of ficolin-1, ficolin-2, or ficolin-3.

### Claim 2 (depends on 1)

The method according to claim 1, wherein renewal capacity and/or pluripotency of the HPC and/or HSC is maintained or increased.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the HSC and/or HPC are further cultured in an environment wherein a concentration of oxygen is less than a normal ambient oxygen concentration.

### Claim 4 (depends on 1)

The method according to claim 1 wherein the HSC and/or HPC are cultured in the presence of at least one of ficolin-1, ficolin-2, or ficolin-3 at a concentration of in the range of approximately 50 ng/ml-200 ng/ml.

### Claim 5 (depends on 1)

The method according to claim 1 wherein the HSC and/or HPC are cultured in the presence of ficolin-1.

### Claim 6 (depends on 3)

The method according to claim 3 wherein the concentration of oxygen is less than approximately 20%, or is less then approximately 12%, or is between approximately of 5%-10% or is approximately 5%.

### Claim 7 (depends on 1)

The method according to claim 1 wherein the HSC and/or HPC are cultured in the presence of growth factors selected from the group comprising interleukins 3, 6 and 11, stem cell factor, stem cell ligand, FLT-3 ligand, and thrombopoietin.

### Claim 8 (depends on 7)

The method according to claim 7 wherein the growth factors are provided at a concentration of about 50 ng/mL-100 ng/mL.

### Claim 9 (depends on 7)

The method according to claim 7 wherein thrombopoietin is provided at about 50 ng/mL, stem cell factor is provided at about 50 ng/mL, Flt-3 ligand is provided at about 80 ng/mL and interleukin 6 is provided at about 100 ng/mL.

### Claim 10 (depends on 1)

The method according to claim 1 wherein the HPC are cultured to increase the number of HPC relative to the initial number of HPCs to produce a second amount of HPCs.

### Claim 11 (depends on 10)

The method according to claim 10, wherein the second amount of HPC are isolated and further cultured in a separate culture medium.

### Claim 12 (depends on 11)

The method according to claim 11, wherein the second amount of HPC are isolated and further cultured in a separate culture medium and the separate culture medium comprises a growth factor selected from the group comprising interleukins 3, 6 and 11, stem cell factor, stem cell ligand, FLT-3 ligand, and thrombopoietin that promotes HPC maintenance, expansion and/or differentiation, to produce differentiated cells of hematopoietic origin.

### Claim 13 (depends on 12)

The method according to claim 12, wherein the separate culture medium includes inoculated stromal cells and/or is a stromal cell conditioned medium.

### Claim 14 (depends on 1)

The method according to claim 1 wherein the HPC are cultured for a period of 6-8 weeks prior to expansion and/or differentiation of HPC and/or HSC.