Conditioned medium from human adult liver stem cells and its use in the treatment of liver disorders

• US 10,874,699 B2
• Filed: 03/29/2018
• Issued: 12/29/2020
• Est. Priority Date: 07/05/2013
• Status: Active Grant

• Current Assignee: Université catholique de Louvain
• Original Assignee: Université catholique de Louvain
• Inventors: Najimi, Mustapha, Sokal, Etienne, Berardis, Silvia
• Primary Examiner(s): Fox, Allison M
• Application Number:
  US15/940,897
• Publication Number:
  US 20180289747A1
• Time in Patent Office: 1,006 Days
• Field of Search: None
• CPC Class Codes: A61K 35/407 Liver; Hepatocytes
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 13/08 of the prostate
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C12N 2502/14 hepatocytes
• CPC Class Codes: C12N 5/0037 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 5/067 Hepatocytes
• CPC Class Codes: C12N 5/0672 Stem cells; Progenitor cells; Precursor cells; Oval cells

Abstract

The invention relates to cell-free compositions obtained by culturing adult-derived human liver stem/progenitor cells (ADHLSC) in cell culture medium and isolating the resulting conditioned medium (ADHLSC-CM) that has advantageous properties, such as anti-fibrotic effects. ADHLSC-CM, compositions based on ADHLSC-CM, and other related and derived products, can be used in cell culture processes or as a medicament, more particularly for the treatment of diseases involving organ injury, organ failure, in organ or cell transplantation or the pathological disruption, inflammation, degeneration, and/or proliferation of cells within a tissue or an organ, in particular within liver.

Claims

1. A method for producing a cell-free conditioned medium comprising the steps of culturing adult-derived human liver stem/progenitor cells (ADHLSC) in a cell culture medium and separating the cell culture medium from the cells, wherein the ADHLSC are albumin-positive, vimentin-positive, alpha smooth muscle actin-positive, cytokeratin-19-negative, and CD133-negative.

2. The method for producing a cell-free conditioned medium according to claim 1, wherein:

(a) the cell culture medium is a serum-free medium; and

/or

(b) the cell culture medium is separated from ADHLSC after culturing ADHLSC in the cell culture medium for at least 2 hours, at least 4 hours, at least 6 hours, at least 8 hours, at least 12 hours, or at least 24 hours; and

/or

(c) the adult-derived human liver stem/progenitor cells (ADHLSC) further express at least one marker selected from CD90, CD73, CD44, CD29, alpha-fetoprotein, alpha-1 antitrypsin, HNF-4 and MRP2 transporter.

3. The method for producing the cell-free conditioned medium of claim 1, wherein the medium comprises Hepatocyte Growth Factor (HGF), Vascular Endothelial Growth Factor (VEGF), Eotaxin (CCL11), Interleukin-6 (IL-6), and Interleukin-8 (IL-8) at a concentration of at least 1 ng/ml comprising the steps of culturing adult-derived human liver stem/progenitor cells (ADHLSC) in a cell culture medium and separating the cell culture medium from the cells.

4. The method of claim 1, wherein the cell-free conditioned medium further comprises micro vesicles.

5. The method of claim 4, wherein the micro vesicles are smaller than 0.40 μ

m.

6. The method of claim 1, wherein the cell-free conditioned medium is concentrated at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 50-fold, or at least about 100-fold.

7. The method of claim 1, wherein the cell-free conditioned medium is diluted at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 50-fold, or at least about 100-fold.

8. The method of claim 1, wherein the cell-free conditioned medium inhibits cell proliferation of hepatic stellate cells (HSCs).

9. The method of claim 1, wherein the cell-free conditioned medium inhibits plating efficiency of HSCs.

10. A method for producing a cell-free composition, comprising:

fractioning the cell-free conditioned medium of claim 1, wherein said fractioning comprises filtering, centrifuging, adsorbing, and/or separating by chromatography the cell-free conditioned medium;

or

enzymatically digesting the cell-free conditioned medium of claim 1.

11. The method of claim 10, wherein a micro vesicle fraction is obtained.