Isolated liver stem cells

• US 9,931,360 B2
• Filed: 08/25/2017
• Issued: 04/03/2018
• Est. Priority Date: 12/21/2005
• Status: Active Grant

• Current Assignee: Université catholique de Louvain
• Original Assignee: Université catholique de Louvain
• Inventors: Sokal, Etienne, Najimi, Mustapha
• Primary Examiner(s): WEHBE, ANNE MARIE SABRINA
• Application Number:
  US15/686,729
• Publication Number:
  US 20170354687A1
• Time in Patent Office: 221 Days
• Field of Search: 435 701, 435370, 435378, 435395, 435402, 435455
• CPC Class Codes: A61K 35/28 Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
• 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 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 3/06 Antihyperlipidemics
• CPC Class Codes: A61P 3/08 for glucose homeostasis pancreatic hormones A61P5/48
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 31/08 for leprosy
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 31/14 for RNA viruses
• CPC Class Codes: A61P 31/20 for DNA viruses
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/04 Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• CPC Class Codes: C12N 5/0672 Stem cells; Progenitor cells; Precursor cells; Oval cells
• CPC Class Codes: G01N 33/5067 Liver cells

Abstract

A method of treating liver-based inborn, metabolic deficiencies is disclosed by treatment of an individual, such as a patient suffering from liver-based inborn, metabolic deficiencies, with human progenitor or stem cells, a cell population or their progeny. The cells used in the treatment have the following characteristics. They are positive for vimentin, α-smooth muscle actin (ASMA), and for at least one mesenchymal marker such as CD90, CD29, CD73, and CD44. They are positive for at least one hepatocyte marker such as albumin, alpha-fetoprotein, alpha-1 antitrypsin, HNF-4 and MRP2 transporter. They express at least one hepatocyte-like property or function such as G6P, CYP1B1, CYP3A4, TDO, TAT, GS, GGT, CK8, and EAAT2. They are negative for at least one marker such as cytokeratin-19, CD45, CD34, CD49f, CD133, HLA-DR, and CD117. They have mesenchymal-like morphology. They originate from human adult liver cells.

Claims

1. A method of producing a protein of interest comprising:

introducing a functional gene encoding a protein of interest into a liver progenitor or stem cell originated from human adult liver,

incubating the population of liver cells under conditions effective for transcription, translation, and optionally post-translational modification to take place, and

harvesting the protein of interest, wherein

(a) the isolated human progenitor or stem cell expresses at least the mesenchymal markers vimentin and α

-smooth muscle actin (ASMA),

(b) the isolated human progenitor or stem cell expresses the hepatocyte marker albumin (ALB),

(c) the isolated human progenitor or stem cell is negative for cytokeratin-19 (CK-19), and

(d) the isolated human progenitor or stem cell has mesenchymal-like morphology.

2. The method of claim 1 in which the protein of interest comprises a vaccine antigen.

3. The method according to claim 1, wherein the isolated human progenitor or stem cell expresses all the markers CD90, CD73, CD44, vimentin and ASMA.

4. The method according to claim 1, wherein the isolated human progenitor or stem cell further expresses one or more other hepatic or hepatocyte markers selected from the group consisting of CD29, alpha-fetoprotein (AFP), alpha-1 antitrypsin, HNF-4 and MRP2 transporter.

5. The method according to claim 4, wherein the isolated human progenitor or stem cell expresses CD29, AFP, alpha-1 antitrypsin and MRP2 transporter.

6. The method according to claim 1, wherein the isolated human progenitor or stem cell is CD90, CD29 and CD44 positive, albumin-positive, vimentin-positive and alpha smooth muscle actin-positive, and negative for CD45, CD34, CD117 and CK-19.

7. The method according to claim 1, wherein the mesenchymal-like morphology, comprises at least one selected from the group consisting of growth in monolayers, flattened form, broad cytoplasm and ovoid nuclei with one or two nucleoli.

8. The method according to claim 1, wherein the isolated human progenitor or stem cell can differentiate into hepatocytes or hepatocyte-like cells and does not differentiate into mesodermal cell types.

9. The method according to claim 1, wherein the isolated human adult liver progenitor or stem cell, corresponds to the cell line as deposited on Feb. 20, 2006 under the Budapest Treaty with the Belgian Coordinated Collections of Microorganisms (BCCM) under accession number LMBP 6452CB, sub-lines thereof including clonal sub-lines, and progeny thereof.

10. A method of producing a protein of interest comprising:

introducing a functional gene encoding a protein of interest into a human progenitor or stem cell, cell population or progeny thereof,

incubating the human progenitor or stem cell, cell population or progeny thereof under conditions effective for transcription, translation, and optionally post-translational modification to take place, and

harvesting the protein of interest, wherein said human progenitor or stem cells, cell population or progeny thereof;

are positive for vimentin, α

-smooth muscle actin (ASMA), and for at least one mesenchymal marker selected from the group consisting of CD90, CD29, CD73, and CD44;

are positive for albumin (ALB);

express at least one molecule indicative of hepatocyte-like properties or function selected from the group consisting of G6P, CYP1B1, CYP3A4, TDO, TAT, GS, GGT, CK8, and EAAT2;

are negative for cytokeratin-19 (CK-19);

have mesenchymal-like morphology; and

originate from human adult liver cells.

11. The method according to claim 10 in which the protein of interest comprises a vaccine antigen.

12. The method according to claim 10, wherein the human progenitor or stem cells, cell population or progeny thereof are positive for vimentin, α

-smooth muscle actin (ASMA), CD90, CD29, CD73, CD44, and albumin (ALB).

13. The method according to claim 10, wherein the human progenitor or stem cells, cell population or progeny thereof are negative for cytokeratin-19 (CK-19), CD45, CD34, CD49f, CD133, HLA-DR, and CD117.

14. The method according to claim 10, wherein the human progenitor or stem cells, cell population or progeny thereof express CYP3A4.

15. The method according to claim 10, wherein the mesenchymal-like morphology comprises at least one selected from the group consisting of growth in monolayers, flattened form, broad cytoplasm and ovoid nuclei with one or two nucleoli.

16. The method according to claim 10, wherein the human progenitor or stem cell line, cell population or progeny thereof, can differentiate into hepatocytes or hepatocyte-like cells and do not differentiate into mesodermal cell types.

17. The method according to claim 10, wherein the functional gene encoding a protein of interest enhances the growth, differentiation and/or functioning of said cells.

18. The method according to claim 10, wherein the functional gene encoding a protein of interest constitutively or inducibly over-express a polypeptide normally expressed by hepatocytes.

19. The method according to claim 10 wherein the human progenitor or stem cells, cell population or progeny thereof are negative for at least one additional marker selected from the group consisting of CD45, CD34, CD49f, CD133, HLA-DR, and CD117.