PROCESS FOR PRODUCING LIVER CELLS

Abstract

The present invention relates to a process for producing liver cells, especially liver stem cells, which after injection into a mammal or in vitro form liver cells that are differentiated, especially differentiated into hepatocytes, into cholangiocytes, and preferably also into liver sinusoidal endothelial cells (LSEC) that can e.g. form blood sinusoidal capillaries. The invention is based on in in vitro producing liver stem cells from a sample of liver tissue, cultivating the liver stem cells in vitro for an increase in cell number. It has been found that the liver stem cells that are produced by the process of the invention can be cultivated and increased in number while maintaining their capability to differentiate into liver cells, especially into hepatocytes, cholangiocytes, and preferably also into LSEC. Accordingly, the process of the invention is suitable for producing liver stem cells that are autologous for the originator of the sample of liver tissue.

Claims

1. A process for producing liver cells, comprising isolating liver cells from a sample of liver tissue to generate primary liver cells and incubating the primary liver cells in cell culture medium containing EGFL6 in order to produce liver cells which are liver stem cells.

2. The process according to claim 1, characterized in that the EGFL6 is contained in the cell culture medium by addition of EGFL6 to the cell culture medium.

3. The process according to claim 2, wherein the primary liver cells are not controlled to contain polynuclear liver cells or are devoid of polynuclear liver cells.

4. The process according to claim 1, wherein the primary liver cells are controlled to contain at least one polynuclear liver cell, that the cell culture medium initially is devoid of added EGFL6, and that incubating is under static conditions for at least 14 days in contact with a gas atmosphere having a lower oxygen concentration than the standard atmosphere and/or a higher CO2 concentration than 5 vol.-% during the entire cultivation period.

5. The process according to claim 1, wherein the primary liver cells are controlled to contain at least one cell that expresses EGFL6.

6. The process according to wherein controlling the primary liver cells to contain at least one polynuclear liver cell comprises analyzing the primary liver cells for presence of polynuclear cells and selecting primary liver cells that contain at least one polynuclear cell.

7. The process according to claim 1, wherein the sample of liver tissue is a sample of human liver tissue.

8. The process according to claim 1, wherein the primary liver cells are provided in the form of a liver biopsy that has been treated by digestion with a protease for at least 6 h under static conditions, and directly incubating the resultant digested liver biopsy in cell culture medium in static culture.

9. The process according to claim 8, wherein prior to the digestion with a protease, the liver biopsy is incubated under static cell culture conditions for at least 6 h up to at least 72 h.

10. The process according to claim 1, wherein the primary liver cells are provided in the form of a liver biopsy, the process comprising directly incubating the liver biopsy in cell culture medium in static culture for at least 6 h under static conditions, followed by treatment of the liver biopsy by digestion with a protease, and subsequently incubating the resultant digested liver biopsy in static culture for at least 6 h under static conditions.

11. The process according to claim 8, wherein the cell culture medium is originally devoid of EGFL6.

12. The process according to claim 8, wherein is added to the cell culture medium at the beginning of incubating the liver biopsy in cell culture medium in static culture or is added during incubating the digested liver biopsy in cell culture medium in static culture.

13. The process to claim 8, comprising after incubating the liver biopsy in cell culture medium in static culture for at least 6 h under static conditions, the treatment of the liver biopsy by digestion with a protease is for 30 min to 2 h.

14. The process according to claim 1, wherein the liver stem cells are in vitro contacted with at least one differentiation factor initiating differentiation into hepatocytes, into cholangiocytes, and/or into liver sinusoidal endothelial cells (LSEC).

15. The process according to claim 1, comprising one of the contacting liver stem cells with an agent in a process for analyzing the effect of the agent onto liver cells.

16. The process according to claim 1, comprising cultivating the liver stem cells for a time that is equivalent to at least 30 passages in static cell culture plates.

17. The process according to claim 1, wherein the liver stem cells are genetically manipulated.

18. The process according to claim 1, comprising injecting the liver stem cells into a non-human mammal having a defective liver for producing a non-human mammal having a liver that is in part or completely comprised of human liver cells.

19. A process for producing liver cells, comprising treating a liver biopsy by digestion with a protease for at least 6 h under static conditions, and directly incubating the resultant digested liver biopsy in cell culture medium in static culture.

20. The process according to claim 19, wherein the cell culture medium is originally devoid of EGFL6.

21. The process according to claim 19, comprising adding EGFL6 to the cell culture medium at the beginning of incubating the digested liver biopsy in cell culture medium in static culture or during incubating the digested liver biopsy in cell culture medium in static culture.

22. The process according to claim 1, comprising injecting a suspension of the liver stem cells into the portal liver vein or into the spleen of an experimental animal having a defective liver.

23. A liver stem cell that expresses the combination of markers EGFL6, KRT18, KRT19, TERT, HNF4 A, FOXA2, and LYVE1.

24. The liver stem cell according to claim 23, wherein the cell expresses CDH2 and CD34.

25-30. (canceled)

Description

[0043] The invention is now described by way of examples and with reference to the figures, which show in

[0044] FIG. 1 graphical representations of analytical results for specific markers of liver stem cells produced according to the invention,

[0045] FIG. 2 a) to f) light micrographs of an exemplary culture of primary human liver cells developing into liver stem cells,

[0046] FIG. 3 a) to c) histological analyses of liver tissue developed from human liver stem cells in mice, and in

[0047] FIG. 4 a light micrograph of an exemplary culture of primary human liver cells developing into liver stem cells.

EXAMPLE 1: PROCESS FOR PRODUCING LIVER STEM CELLS FROM PRIMARY HUMAN LIVER CELLS

[0048] Primary liver cells were prepared from human liver tissue that was received from liver surgery. Prior analysis had shown that the liver tissue was no tumour tissue. The liver tissue was digested by collagenase treatment using 0.05% collagenase P in pre-warmed digestion buffer (DB) at 37° C. for 15-20 min, followed by further mechanical disruption and pouring of the emerging cell suspension through a gauze-lined funnel and centrifuged (50×g, 5 min, 4° C.). The isolated cells, now regarded as primary liver cells, were seeded at a density of at least 400 000 cells in culture dishes (2.2 cm diameter) that were non-coated plastic, optionally coated with Collagen I, in cell culture medium. The medium could be Williams E, RPMI 1640, DMEM(:F12), Promocell Hepatocyte Growth Medium, preferably hepatocyte maintenance medium Lonza, e.g. HCM. Generally preferable, the medium contained human serum, e.g. 1-10 Vol. %.

[0049] Cultivation was static at 37° C. in a 5% CO.sub.2 atmosphere, preferably the circumferential gas vents of these static culture dishes were closed by adhesive tape to leave an exchange cross-section of only 1/10.sup.th to 1/20.sup.th of the total circumference. Cultivation was for 4 to 8 days without movement before removing the dishes from the incubator to check cells under a microscope. The medium was not exchanged during the initial 29 days of the culture.

[0050] FIG. 1 shows light microscopic pictures (size bar is 250 μm) of the same section of one exemplary culture dish, a) at day 14, b) at day 17, c) at day 28, d) at day 23, e) at day 26, and f) at day 29 after seeding. This shows the albeit slow occurrence of optically differing cell clusters. Cells of these clusters have been characterized later as being EGFL6.sup.+-liver stem cells. These liver stem cells could be propagated, e.g. when reaching 90% confluence or more, with trypsination and passaging to further dishes, using the same fresh medium, again under static culture conditions.

EXAMPLE 2: PROCESS FOR PRODUCING LIVER STEM CELLS FROM PRIMARY HUMAN LIVER CELLS

[0051] In accordance with the preferred embodiment, primary liver cells that were isolated from a human liver tissue sample were incubated in medium containing added human EGFL6 (obtained from Sino Biological), e.g. at a concentration of 200 ng/ml.

[0052] Cultivation of primary liver cells in medium containing EGFL6 resulted in the production of liver stem cells both from polynuclear liver cells, and from mononuclear liver cells. The occurrence of clusters of liver stem cells in static culture therefore was possible for all primary liver cells, and it was more efficient than in the process using cultivation without added EGFL6.

[0053] Passaging of liver stem cells, e.g. when reaching up to 90% confluence, to-date is at least 40 passages, resulting in an increase 2-10 fold/passage of the number of liver stem cells.

[0054] The liver stem cells produced by the process were analysed by reverse-transcription quantitative PCR (RT-qPCR). In short, total mRNA was isolated from cultivated liver stem cells using RNeasy Plus micro Kit (obtained from Qiagen), and mRNA was analysed by RT-qPCR using primer pairs specific for markers as indicated in FIG. 2, which shows the results. In FIG. 1, mean quantities of the specific amplificates and standard deviations are indicated for liver stem cells that were generated from liver tissue samples obtained from four different patients, all non-cancer patients.

[0055] The result shows that the EGFL6-expressing liver stem cells produced by the process of the invention express markers for hepatocytes, for cholangiocytes and for LSEC, which in their combination and with expression of signalling receptors are also regarded as stem cells of endodermal origin in developmental terms. In detail, for liver stem cells according to the invention robust transcript levels for ALB, TDO, HNF4 A, CYP3 A4, KRT8, KRT18 and FAH indicated hepatocytes; SLC4 A2 (AE2), SCTR, OPN, KRT7, KRT19, CFTR, GGT indicated cholangiocytes; VWF, F8, FCGR2B (CD32b), LYVE1, SELE, KDR, FLT4 indicated LSEC; NCAM1, PKM, SALL4, SOX17, MCAM, TERT, THY1 (CD90) indicated stem cells; TNFRSF1 A, NOTCH2, PTCH1, CTNNB1 (b-Catenin), YEP1, RYK, LGR5 are signalling receptors indicating response potential for the particular signalling pathways; and CEBPA, CEBPB, FOXA2, together with HNF4 A are transcription factors also indicating endodermal origin; CDH1 (E-Cad), CDH2 (N-Cad), EPCAM, TWF1, are cadherins and adhesion molecules indicating both epithelial as well as mesenchymal cell potential.

EXAMPLE 3: PROCESS FOR GENERATING AN EXPERIMENTAL ANIMAL CONTAINING LIVER TISSUE GENERATED FROM HUMAN LIVER STEM CELLS

[0056] The process for generating an experimental animal containing human liver tissue also exemplifies the use of the liver stem cells for treatment of liver defects in human patients. For use in human patients, the liver stem cells are preferably generated from a liver tissue sample obtained from the patient, who is the later recipient of the liver stem cells, and therefore the liver stem cells are autologous cells that are immunologically compatible to the patient.

[0057] Human liver stem cells produced according to Example 2 and as an example for genetic in vitro manipulation were transduced with a lentiviral nucleic acid construct encoding GFP under the control of a SFFV promoter.

[0058] These genetically modified human liver stem cells were introduced by injection into the spleen or into the liver portal vein of two strains of immune deficient mice, Rag2−/− and γc−/− mouse (RAG mouse), or a FAH−/− and Rag2−/− and γc−/− mouse (FRG mouse), 5 to 4 mice each, total of 19 mice. FAH−/− mice had a chronically defective liver due to withdrawal of the drug NTBC after cell transplantation. After collecting the human liver stem cells from the culture plate, they were resuspended in PBS, and then were intrasplenically injected at a quantity of ca. 500 000 cells/mouse, using a wide gauge needle. All animal care regulations and anaesthesia were applied.

[0059] The RAG mice were kept with standard laboratory mouse diet. FRG mice were kept with standard laboratory diet but drinking water was supplemented with NTBC before injection of liver stem cells and drinking water without NTBC after injection of liver stem cells, in order to induce chronic cell damage in the endogenous murine liver and, after injection of the human liver stem cells, promoting growth of the injected human cells.

[0060] After about 90 days, mice were killed and fully examined. It was found that no liver cells were present outside of the normal location of liver, and the liver was partially or completely made up of human cells.

[0061] FIG. 3 a) to i) shows micrographs of mouse liver sections, in FIG. 3 a), b) and c) of an exemplary FRG mouse and in FIG. 3 d), e) and f) for an exemplary Rag2−/− γc−/− mouse. Immune histochemical staining in a), d) and g) was by HRP/DAPI, immune fluorescence staining in b), e) and h) was by FITC. Immune staining for human Albumin using an anti-human albumin antibody (α-hALB, obtained from Bethyl Laboratories) is shown in c) for a FRG mouse, and in f) for a Rag2−/− γc−/− mouse. FIG. 3 i) shows immune staining for GFP using an anti-GFP antibody (α-GFP, obtained from Abcam) in the liver of a FRG mouse.

[0062] These results show that the human liver stem cells made up at least part of the liver tissue in the mouse, and specifically, the human liver stem cells generated non-hepatocytic LSEC-networks as indicated by staining for human Albumin (hALB-positive), indicating LSEC originating from the human liver stem cells. The liver sections of the FRG mouse in immune staining for GFP indicate whole bile ducts generated from the human liver stem cells. The immune staining via HRP/DAPI immunohistochemistry shows the lining of a bile duct by individual human cholangiocytes generated from the human liver stem cells.

EXAMPLE 4: PROCESS FOR PRODUCING LIVER STEM CELLS FROM HUMAN LIVER TISSUE

[0063] Human liver tissue biopsy samples, approximate circular cross-section of 5 mm, 10 mm length or cut to 5 mm length, were obtained from surgery patients and used on different days when the biopsy became available. Within 30 to 60 min after withdrawal of the biopsy, these biopsy samples were submerged in 10 mL of a 5 mg/10 mL collagenase solution in digestion buffer (DB) containing 58 mM NaCl, 5.8 mM KCl, 0.5 mM CaCl.sub.22H.sub.2O, 100 mM Hepes and 0.5% Albumin at pH7.6, and incubated at 37° C. under static conditions for 6 h, 12 h, 18 h or 24 h, up to 72 h, or up to 10 d or up to 7 d. Initial experiments, in which the protease treatment of a biopsy was for 1 h under otherwise identical conditions are regarded as a comparative example only, because subsequent incubation under cell culture conditions in medium devoid of EGFL6 did not yield occurrence of liver stem cells at day 7 of the cultivation.

[0064] As a representative result, FIG. 4 shows a micrograph of cells that were produced when incubating a liver biopsy that was treated for 12 h by the protease in DB at 37° C. under static conditions and immediately subsequently transferred into cell culture medium devoid of EGFL6 and incubated under static conditions. The micrograph shows the occurrence of cells having the typical morphology of liver stem cells on day 7 of the static incubation. The liver stem cells are marked-up by the handwritten line. These cells were identified as liver stem cells according to the invention by detection of their specific markers.

[0065] The production of liver stem cells was confirmed for liver biopsies treated by 6 h, 8 h, up to 18 or up to 24 h, 48 h, 72 h or at least 72 h of protease digestion at 37° C. under static conditions. Optionally, the culture medium was supplemented with EGFL6, e.g. from the beginning of the cultivation or during the cultivation, e.g. on day 2 or day 3. When using culture medium with added EGFL6, growth of liver stem cells was found to be faster than when using medium without adding EGFL6.

EXAMPLE 5: PROCESS FOR PRODUCING LIVER STEM CELLS FROM HUMAN LIVER TISSUE

[0066] As a variation of Example 4, human liver tissue biopsy samples, approximate circular cross-section of 5 mm, 10 mm length or cut to 5 mm length, obtained from surgery patients, were first incubated in culture medium under static cell culture conditions without added EGFL6 for 6 h up to 10 d and thereafter were treated by collagenase digestion to generate a digested liver biopsy, For the protease digestion, subsequent to the initial incubation under static cell culture conditions, the biopsy samples were submerged in 10 mL of a 5 mg/10 mL collagenase solution in DB for 30 min to at least 1 h. The resultant digested biopsy samples were incubated at 37° C. under static cultivation conditions in culture medium without added EGFL6, or in the same medium but with EGFL6 added.

[0067] It was found that cells that were produced when treating a liver biopsy shows the occurrence of cells having the typical morphology of liver stem cells on day 5 to 7 of the static incubation that followed the protease digestion, both in medium without EGFL6 added, and more cells in medium with EGFL6 added. These cells were identified as liver stem cells according to the invention by detection of their specific markers.