|Número de publicación||WO2013068557 A1|
|Tipo de publicación||Solicitud|
|Número de solicitud||PCT/EP2012/072316|
|Fecha de publicación||16 May 2013|
|Fecha de presentación||9 Nov 2012|
|Fecha de prioridad||9 Nov 2011|
|Número de publicación||PCT/2012/72316, PCT/EP/12/072316, PCT/EP/12/72316, PCT/EP/2012/072316, PCT/EP/2012/72316, PCT/EP12/072316, PCT/EP12/72316, PCT/EP12072316, PCT/EP1272316, PCT/EP2012/072316, PCT/EP2012/72316, PCT/EP2012072316, PCT/EP201272316, WO 2013/068557 A1, WO 2013068557 A1, WO 2013068557A1, WO-A1-2013068557, WO2013/068557A1, WO2013068557 A1, WO2013068557A1|
|Inventores||Frederik NEVENS, Johan Neyts, Jan Paeshuyse, Philip ROELANDT, Cathérine VERFAILLIE|
|Solicitante||Katholieke Universiteit Leuven|
|Exportar cita||BiBTeX, EndNote, RefMan|
|Citas de patentes (44), Otras citas (80), Citada por (1), Clasificaciones (15), Eventos legales (3)|
|Enlaces externos: Patentscope, Espacenet|
VIRUS INFECTABLE STEM CELLS
FIELD OF THE INVENTION
The invention is directed to methods for culturing cells so that the cells are induced to differentiate into cells that express a hepatocyte phenotype and/or hepatocyte progenitor phenotype which support complete replication of hepatitis viruses and other hepatotropic pathogens, more particularly hepatitis C virus (HCV). More particularly, the invention relates to methods for culturing cells so that the cells are induced to differentiate into cells that express a definitive endodermal phenotype, a liver-committed endodermal phenotype, a hepatoblast phenotype, and hepatocyte phenotype which can be infected with and support the complete replication of hepatitis viruses, more particularly hepatitis C virus. The invention relates to the development of a tool suitable for the search, discovery and validation of inhibitors of HCV replication and of other hepatotropic pathogens, more particularly HCV antiviral drugs and therapies. The invention further relates to methods for inducing hepatotropic virus, replication in vitro, and more particularly to a simple in vitro replication assay of hepatotropic viruses which enables productive and sustained infectious hepatotropic virus production, eg. for the hepatotropic virus HCV. The cells produced by the methods of the invention are thus useful, among other things, for toxicity studies and screening for treatments/drugs against hepatitis viruses, more particularly hepatitis C virus.
BACKGROUND OF THE INVENTION The scarcity of primary human hepatocytes generated a significant interest in the production of human stem cell-derived functionally active hepatocyte-like cells. The development of induced pluripotent stem cells (iPSC) offer the possibility of generating patient-specific stem cells and progeny. Worldwide about 180 million people are chronically infected with the hepatitis C virus (HCV). The virus is the leading cause of chronic liver disease in the Western world. When left untreated, patients have a high chance of developing cirrhosis and hepatocellular carcinoma. Treatment with pegylated interferon and ribavirin for 24 to 48 weeks, the current standard of care, clears the virus in about 50% of the patients but is most often associated by hematological, systemic and psychological side effects. The HCV NS3 protease inhibitors, boceprevir and telaprevir, have recently been approved for use in combination with the standard of care. A number of other potent antivirals are currently in clinical development.
Culture systems for HCV are available [using (sub)genomic replicons and infectious virus] and make mainly use of hepatoma cells (Zhong, J., et al. 2005). However, metabolic differences between rapidly proliferating cancer cell lines and quiescent primary cells, as well as mutations in the cell line dsR A sensor retinoic acid-inducible gene-I (RIG-I) (Bartenschlager, R. & Pietschmann, 2005), suggest that this culture model fails to capture many aspects of HCV infection in patients. Alternatively, primary human hepatocytes were shown by several groups to support HCV infection in vitro. Immunodeficient mice repopulated with human hepatocytes, were shown to support HCV infection in vivo (Meuleman, P. & Leroux-Roels, G. 2008). However, the last two models are limited because shortage of human livers to isolate hepatocytes. Since mature functioning hepatocytes cannot be readily expanded in culture, other cell sources should be considered as an alternative model.
Therefore, there is still a huge need for easily expandable functionally active hepatocytes or hepatocyte-like cells, which are infectable and which support the complete replication of hepatotropic pathogens such as hepatitis C virus. The present invention fulfills these needs by demonstrating that hepatocytes derived from stem cells, more particularly human embryonic stem cells (hESC) (H9 and HI cells) or hiPSC, can support the complete HCV replication cycle including the production of infectious virus. Furthermore, similar results were obtained for other hepatotropic pathogens such as Hepatitis A Virus, Dengue Fever Virus and Yellow Fever Virus.
SUMMARY OF THE INVENTION
The invention is based on methods developed by the inventors to produce a renewable source of hepatocytes in vitro, which is infectable with hepatitis virus and supports the complete replication of hepatitis viruses, more particularly HCV. Said renewable source of hepatocytes, the method of the present invention and the screening assays disclosed herein, are useful for the screening and development of compounds, drugs and therapies for diseases caused by hepatotropic pathogens, such as viral hepatotropic pathogens, more particularly Hepatitis viruses such as HAV and HCV; and Flaviviruses such as Dengue Fever Virus and Yellow Fever Virus. Cell culture conditions were developed in view of gene expression and optimized from conditions that were previously developed by the inventors (Roelandt, P., et al. 2010, WO2010049752 and WO2011158125). WO2009013254 also discloses methods for inducing human blastocysts stem cells to differentiate into hepatocyte-like cell progenitor cells. Said prior art methods, however, have the disadvantage that multiple expensive growth factors are used in each step of the differentiation protocol. Furthermore, none of said prior art documents actually discloses the infection of the obtained cells with hepatotropic pathogens.
The present invention aimed at further optimizing specific cell culture conditions to successfully produce cells that express phenotypes of hepatocytes which were subsequently successfully infected with hepatotropic pathogens, and which furthermore were able to support the complete replication of hepatotropic viruses. As evident from the examples disclosed herein, the newly optimized culture method according to the present invention, is in particular interesting in that the cells can be grown FBS-free and in the presence of less growth factors.
Numbered statements of the invention are as follows:
1. A method for inducing stem cells to differentiate into hepatocyte cells or cells with a hepatocyte phenotype, said method comprising the steps of:
(a) culturing stem cells in ActivinA containing medium
(b) then culturing the cells of step (a) in BMP4 containing medium
(c) then culturing the cells of step (b) in FGF1 containing medium; and
(d) then culturing the cells of step (c) in HGF containing medium;
wherein at least in one of steps (a), (b) or (c) only a single growth factor is used.
2. The method according to statement 1, wherein
in step (a) the ActivinA concentration in the medium is from about 10 ng/ml to about 1000 ng/ml; in particular about 100 ng/ml;
in step (b) the BMP4 concentration in the medium is from about 5 ng/ml to about 500 ng/ml; in particular about 50 ng/ml;
in step (c) the FGF1 concentration in the medium is from about 5 ng/ml to about 500 ng/ml; in particular about 50 ng/ml; and in step (d) the HGF concentration in the medium is from about 2 ng/ml to about 200 ng/ml; in particular about 20 ng/ml.
3. The method according to anyone of statements 1 or 2, wherein the Activin A containing medium of step (a) further comprises Wnt3a.
4. The method according to statement 3, wherein the Wnt3a concentration is from about 5 ng/ml to about 500 ng/ml; in particular about 50 ng/ml. 5. The method according to anyone of statements 1 to 4, wherein in one or more of steps (a), (b), (c) or (d), the cells are cultured for at least four days.
6. The method according to anyone of statements 1 to 5, wherein the cells are cultured for about four to seven days in step (a), in particular about four days;
about four to seven days in step (b), in particular about four days;
about four to seven days in step (c), in particular about four days and
about four to thirty days in step (d), in particular about sixteen days.
7. The method according to anyone of statements 1 to 6, wherein step (a) comprises culturing the cells for about two days in ActivinA- and Wnt3a-containing medium followed by culturing for about two days in ActivinA containing medium.
8. The method according to statement 1, comprising to steps of:
(a) culturing stem cells with 100 ng/ml Activin-A and 50 ng/ml Wnt3a for 2 days, and subsequently culturing the cells with 100 ng/ml Activin-A for 2 more days;
(b) then culturing the cells of step (a) with 50 ng/ml BMP4 for 4 days;
(c) then culturing the cells of step (b) with 50 ng/ml FGF1 for 4 days; and
(d) then culturing the cells of step (c) with 20 ng/ml HGF for 16 days. 9. The method according to anyone of statements 1 to 8, wherein in one or more steps, the cells are cultured in the absence of serum. 10. The method according to anyone of statements 1 to 9, wherein the stem cells are Embyronic Stem Cells (ESC) or induced Pluripotent Stem Cells (iPSC).
11. The method according to anyone of statements 1 to 10, wherein the stem cells are human.
12. Use of stem cell derived hepatocytes obtainable by the method according to anyone of statements 1-12 as an in vitro culturing system of hepatotropic pathogen.
13. Use of stem cell derived hepatocytes obtainable by the method according to anyone of statements 1-12 for toxicity screening and/or drug testing.
14. Use according to anyone of statements 12 and 13 wherein the hepatotropic pathogen is a hepatotropic virus. 15. Use according to anyone of statements 12 and 13 for the screening and development of compounds, drugs and therapies for hepatotropic pathogens.
16. Use according to statement 15, for the screening and development of compounds, drugs and therapies for essentially all the different stages of virus replication, in particular virus entry, replication comprising (-) and (+) strand synthesis, viral protein synthesis, virus assembly, virus trafficking, or virus release.
17. Use according to statement 14, wherein the hepatotropic virus is selected from: Yellow Fever Virus, Dengue Fever Virus, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D, Hepatitis E, and Hepatitis G virus; in particular hepatitis C.
18. A screening assay for agents which possesses anti-viral activity, comprising:
(a) culturing differentiated ESC or differentiated iPSC in the presence of a virus and said agent; and
(b) assaying replication, translation, assembly infection or the like of said virus. 19. A screening assay for agents which possesses anti-hepatotropic pathogen activity, comprising:
(a) culturing hepatocyte differentiated ESC or hepatocyte differentiated iPSC in the presence of a hepatotropic pathogen and said agent; and
(b) assaying replication, translation, assembly infection or the like of said hepatotropic pathogen.
20. A screening assay for agents which possesses anti-hepatotropic pathogen activity, comprising:
(a) inducing stem cells according to the method as defined in anyone of statements 1-10;
(b) culturing the cells obtained in step (a) in the presence of a hepatotropic pathogen and said agent; and
(c) assaying replication, translation, assembly infection or the like of said hepatotropic pathogen.
21. A screening assay for agents which possesses anti -hepatitis activity, comprising:
(a) inducing stem cells according to the method as defined in anyone of statements 1-10;
(b) culturing the cells obtained in step (a) in the presence of a hepatitis virus and said agent; and
(c) assaying replication, translation, assembly infection or the like of said hepatitis virus.
22. The assay according to statement 21, wherein the hepatitis virus is HCV.
23. A method for identifying an agent with anti-hepatotropic pathogen activity, comprising:
(a) providing a screening assay according to anyone of statements 19-20; and
(b) detecting if said test compound inhibits the biological activity of said hepatotropic pathogen; wherein a test agent which inhibits said biological activity is a compound with said inhibitory effect.
24. The method of statement 23, wherein the agent is a compound selected from a library of compounds, wherein the selected compound with said inhibitory effect is further modified by medicinal chemistry to provide further analogs of said selected compound also having said inhibitory effect. 25. An anti-hepatotropic agent identified by making use of the screening assay according to anyone of statements 18-21 or the method according to anyone of statements 23-24. 26. A pharmaceutical composition for the treatment of an hepatotropic infection, wherein said composition comprises an anti-hepatotropic agent according to statement 25.
Further numbered statements of the invention are as follows: 27. An in vitro culture system for a hepatotropic virus which enables the screening and development of compounds, drugs and therapies for hepatotropic viral diseases, comprising:
(a) culturing stem cells in ActivinA containing medium;
(b) then culturing the cells of step (a) in BMP4 containing medium;
(c) then culturing the cells of step (b) in FGF1 containing medium; and
(d) then culturing the cells of step (c) in HGF containing medium.
28. The system of statement 27, which enables the screening and development of compounds, drugs and therapies for essentially all the different stages of virus replication such as virus entry, replication comprising (-) and (+) strand synthesis, viral protein synthesis, virus assembly, virus trafficking, and virus release.
29. The system of statement 27 and 28, wherein the hepatotropic virus is selected from: Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D, Hepatitis E, Hepatitis F, and Hepatitis G virus.
30. The system of statement 27 to 29, wherein the hepatotropic virus is HCV.
31. The system of statement 27 to 30, wherein the cells are differentiated towards hepatocytes, further comprising culturing the cells in additional differentiation factors.
32. The system of statement 27 to 31, wherein in step (a) the ActivinA concentration in the medium is about 100 ng/ml; in step (b) the BMP4 concentration in the medium is about 50 ng/ml; in step (c) the FGF1 concentration in the medium is about 50 ng/ml; and in step (d) the HGF concentration in the medium is about 20 ng/ml.
33. The system of statement 27 to 32, wherein the Activin A containing medium of step (a) further comprises Wnt3a.
34. The system of statement 33, wherein the Wnt3a concentration is about 50 ng/ml.
35. The system of statement 27 to 34, wherein the cells are cultured at one or more steps for at least four days.
36. The system of statement 27 to 35, wherein the cells are cultured for about four days in step (a), about four days in step (b), about four days in step (c), and about sixteen days in step (d).
37. The system of statement 25 to 36, wherein step (a) comprises culturing the cells for about two days in ActivinA- and Wnt3a-containing medium followed by culturing for about two days in ActivinA containing medium. 38. The system of statement 27 to 37, wherein the cells are differentiated with the differentiation protocol, comprising:
culturing stem cells with 100 ng/ml Activin- A and 50 ng/ml Wnt3a for 2 days, and subsequently culturing the cells with 100 ng/ml Activin-A for 2 more days;
then culturing the cells of step (a) with 50 ng/ml BMP4 for 4 days;
then culturing the cells of step (b) with 50 ng/ml FGF1 for 4 days; and
then culturing the cells of step (c) with 20 ng/ml HGF for 16 days.
39. The system of statement 27 to 38, wherein the cells are cultured at one or more steps in the absence of serum.
40. The system of statement 27 to 39, wherein the stem cells are ESC or iPSC.
41. The system of statement 27 to 40, wherein the stem cells are human. 42. An assay for screening a test agent and selecting an agent which possesses anti-hepatitis activity, comprising:
growing a hepatotropic virus infected cell according to an in vitro culture system of the present invention; and
assaying replication, translation, assembly infection or the like of said hepatotropic virus.
43. The assay of statement 18, wherein the hepatotropic virus is HCV. 44. A method for identifying a compound with anti hepatitis virus activity, comprising:
providing a screening assay comprising a measurable biological activity of a hepatotropic virus in an in vitro culture system of the present invention;
contacting said screening assay with a test compound; and
detecting if said test compound inhibits the biological activity of said hepatotropic virus; wherein a test compound which inhibits said biological activity is a compound with said inhibitory effect.
45. The method of statement 44, wherein the screening assay is the assay of statement 42 or 43.
46. The method of statement 44 or 45, wherein the compound is selected from a library of compounds, wherein the selected compound with said inhibitory or therapeutic effect is further modified by medicinal chemistry to provide further analogs of said selected compound also having said therapeutic effect.
47. A compound having therapeutic effect on a hepatotropic virus, comprising:
(a) providing a screening assay comprising a measurable biological activity of said hepatotropic virus;
(b) contacting said screening assay with a test compound; and
(c) detecting if said test compound inhibits the biological activity of said hepatotropic virus; wherein a test compound which inhibits said biological activity is a compound with said inhibitory or therapeutic effect. 48. The compound of statement 47, wherein the hepatotropic virus is HCV.
49. The compound of statement 47 or 48, wherein the compound is selected from a library of compounds, wherein the selected compound with said therapeutic effect is further modified by medicinal chemistry to provide further analogs of said selected compound also having said therapeutic effect.
50. A pharmaceutical composition for the treatment of a hepatotropic infection, wherein said composition comprises the compound of statement 27 to 49.
Any stem cell can be used in the embodiments of this invention as long as it can be differentiated towards a hepatocyte cell or a cell with a hepatocyte phenotype. Particular differentiation protocols and methods for differentiation of such stem cells are well known to a person skilled in the art and are described for example in WO2010049752.
Specific differentiation methods and protocols for embryonic stem cells (ESC) and for reprogrammed somatic cells (IPSC) are described in the present invention. In one embodiment, the stem cells are embryonic stem cells, more particularly, established embryonic stem cell lines may be used as ESC in the present invention. In another embodiment, the stem cells are iPSC.
The invention provides a method for inducing stem cells to differentiate into hepatocyte cells or cells with a hepatocyte phenotype (i.e. in vitro culture system), infectable with a hepatotropic pathogen. It further provides a screening assay for identification and testing compounds, drugs and therapies for their therapeutic effect on the disease caused by said hepatotropic pathogen.
The invention is, next to a screening tool or assay, also directed to methods of using the differentiated stem cells for studies of liver toxicity, for example, to identify or assess the toxicity of specific compounds. In a particular embodiment, the invention provides the means to assess for sensitivity or resistance of a particular hepatotropic pathogen, to a known antiviral compound or candidate antiviral compound. In a related embodiment, such assessment enables an adaptation of the therapeutic regimen to better suit the sensitivity profile of the particular hepatotropic pathogen.
In one embodiment of the present invention, the hepatotropic pathogen is a viral hepatotropic pathogen or a hepatotropic virus such as a hepatitis virus or a flavivirus. In another embodiment of the present invention, the hepatotropic pathogen is a non-viral or other hepatotropic pathogen such as a parasitic pathogen, such as for example Plasmodium.
In one embodiment of the present invention, the hepatotropic virus is a flavivirus such as Dengue Fever virus or Yellow Fever Virus; or a hepatitis virus such as hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis F, or hepatitis G virus (also known as GB virus C). In a particular embodiment of this invention, the hepatotropic virus is hepatitis C virus (HCV).
In one embodiment of the present invention, hepatitis is hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis F, or hepatitis G. In a particular embodiment of this invention, said hepatitis is hepatitis C.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1. HCV entry. RT-qPCR analysis of gene expression in the differentiated hESC-H9 progeny that were used to be inoculated with HCV (day 20) compared to gene expression in primary hepatocytes (n=3).
Figure 2. Replication and egress of HCV. (A) Quantification of HCV positive strand RNA (by RT-qPCR) in (i) intracellular extracts, supernatants of (ii) human stem cell (hSC) progeny and (iii) Huh7.5.1 cells 10 days after HCV inoculation (n=4). (B) Levels of albumin, ApoBlOO and HCV core protein in supernatants of hSC-progeny at different time points after inoculation (n=3). (E) Infection of Huh7.5.1 with mock (a), HCV (b), supernatants collected 6 days post-infection of hSC-progeny with HCV (c), 10 days post-infection (d) and 13 days post-infection (e). Representative for n=2. Size bar 10 μηι.
Figure 3. Modifications to the published protocol (Roelandt, P., et al. 2010): Activin-A & Wnt3a dO-2, Activin-A d2-4, BMP4 d4-8, FGFl d8-12, HGF dl2-28, no fetal bovine serum (A) Relative gene expression between initial and optimized protocol (B, C, D) Comparison of functional properties of hESC-progeny on day 28 between initial and optimized protocol.
Figure 4. Inhibition of HCV Replication. HCV RNA replication in hESC-progeny (i) untreated (6.2 + 1.6 fg), (ii) treated with 10 μΜ HCV-796 and (iii) treated with 1 μΜ VX-950 (n=2). Figure 5. (A) Levels of HAV RNA in culture supernatant of stem cell-derived hepatocytes in culture supernatant at different days post infection and (B) intracellularly at day 31 post infection.
DETAILED DESCRIPTION OF THE INVENTION Definitions
"A" or "an" means one or more than one.
"Comprising" means, without other limitation, including the referent, necessarily, without any qualification or exclusion on what else may be included. For example, "a composition comprising x and y" encompasses any composition that contains x and y, no matter what other components may be present in the composition. Likewise, "a method comprising the step of x" encompasses any method in which x is carried out, whether x is the only step in the method or it is only one of the steps, no matter how many other steps there may be and no matter how simple or complex x is in comparison to them. "Comprised of and similar phrases using words of the root "comprise" are used herein as synonyms of "comprising" and have the same meaning.
"Effective amount" generally means an amount which provides the desired local or systemic effect. For example, an effective amount is an amount sufficient to effectuate a beneficial or desired clinical result. The effective amounts can be provided all at once in a single administration or in fractional amounts that provide the effective amount in several administrations. The precise determination of what would be considered an effective amount may be based on factors individual to each subject, including their size, age, injury, and/or disease or injury being treated, and amount of time since the injury occurred or the disease began. One skilled in the art will be able to determine the effective amount for a given subject based on these considerations which are routine in the art. As used herein, "effective dose" means the same as "effective amount."
"EC cells" were discovered from analysis of a type of cancer called a teratocarcinoma. In 1964, researchers noted that a single cell in teratocarcinomas could be isolated and remain undifferentiated in culture. This type of stem cell became known as an embryonic carcinoma cell (EC cell).
"Embryonic Stem Cells (ESC)" are well known in the art and have been prepared from many different mammalian species for many years. Embryonic stem cells are stem cells derived from the inner cell mass of an early stage embryo known as a blastocyst. They are able to differentiate into all derivatives of the three primary germ layers: ectoderm, endoderm, and mesoderm. These include each of the more than 220 cell types in the adult body. The ES cells can become any tissue in the body, excluding placenta. Only the morula's cells are totipotent, able to become all tissues and a placenta. hESC are embryonic stem cells of human origing and several established hESC cell lines exist such as the ESC cell lines hESC-H9 and hESC- Hl .
"Hepatic differentiation factors" are chemical or biological factors that induce differentiation of stem and progenitor cells into more differentiated cells of the hepatic lineage. Hepatic differentiation factors include, but are not limited to, Wnt3a, Activin-A, FGF2, BMP4, FGF1, FGF4, FGF8b, HGF and Follistatin. The initial cell may express Oct3/4.
"Hepatocytes" are cells with a hepathic phenotype.
"Hepatocyte phenotype" is a particular phenotype of cells that express albumin and keratin 18 (K T18) but not alpha fetoprotein (AFP) and keratin 19 (K T19); in addition, hepatocytes may express one or more of ALB, TAT, MRP2, G6P, PEPCK1, AAT, CX32, NTCP, OATP1B1, F5, F7, F9, VKORC1, CYP7A1 and CYP3A4. "Hepatotropic pathogens" include two groups of pathogens:
- viral pathogens or hepatotropic viruses such as, but not limited to, hepatitis A, B, C, D, E, F and G virus; human cytomegalovirus (HCMV); Epstein-Barr virus (EBV); herpes simplex virus 1 & 2 (HSV-1 & HSV-2); varicella zoster virus (VZV); human herpesvirus type 6 (HHV6); measles virus; Rubella virus; enteroviruses; yellow fever virus; dengue fever virus; Crimean Congo hemorrhagic fever virus; Arenaviruses, including but not limited to, Junin virus (causing Argentinian hemorrhagic fever), Machupo virus (causing Bolivian hemorrhagic fever), Lassa virus (causing Lassa fever); Rift Valley fever virus; Ebola virus and Marburg virus; and - parasitic pathogens such as, but not limited to, Plasmodium; Echinococcosis;
Fascioliasis; Amebic liver disease causing pathogens such as, but not limited to, Entamoeba histolytica; Fungal abscess causing pathogens such as, but not limited to, the Candida species; Pyogenic liver abscess causing pathogens or bacteria such as, but not limited to, Streptococcus, E. coli, Klebsiella, Proteus, and Staphylococcus. A hepatotropic infection is an infection with a hepatotropic pathogen.
"Human stem cell" or "hSC" is a stem cell as defined in this specification, from human origin. This includes human Embryonic Stem Cells and human Induced pluripotent stem cells.
Use of the term "includes" is not intended to be limiting. For example, stating that an inhibitor "includes fragments and variants does not mean that other forms of the inhibitor are excluded.
"Induced pluripotent stem cells (IPSC or IPS cells)" are somatic cells that have been reprogrammed. for example, by introducing exogenous genes that confer on the somatic cell a less differentiated phenotype. These cells can then be induced to differentiate into less differentiated progeny. IPS cells have been derived using modifications of an approach originally discovered in 2006 (Yamanaka, S. et al, Cell Stem Cell, 1 :39-49 (2007)). For example, in one instance, to create IPS cells, scientists started with skin cells that were then modified by a standard laboratory technique using retroviruses to insert genes into the cellular DNA. In one instance, the inserted genes were Oct4, Sox2, Lif4, and c-myc, known to act together as natural regulators to keep cells in an embryonic stem cell-like state. These cells have been described in the literature. See, for example, Wernig et al, PNAS, 105:5856- 5861 (2008); Jaenisch et al, Cell, 132:567-582 (2008); Hanna et al, Cell, 133:250-264 (2008); and Brambrink et al, Cell Stem Cell, 2: 151-159 (2008). These references are incorporated by reference for teaching IPSCs and methods for producing them. It is also possible that such cells can be created by specific culture conditions (exposure to specific agents).
The term "isolated" refers to a cell or cells that are not associated with one or more cells or one or more cellular components that are associated with the cell or cells in vivo. An "enriched population" means a relative increase in numbers of a desired cell relative to one or more other cell types in vivo or in primary culture.
However, as used herein, the term "isolated" does not indicate the presence of only a specific desired cell, such as a stem or hepatic progenitor cell. Rather, the term "isolated" indicates that the cells are removed from their natural tissue environment and are present at a higher concentration as compared to the normal tissue environment. Accordingly, an "isolated" cell population may further include cell types in addition to stem cells and may include additional tissue components. This also can be expressed in terms of cell doublings, for examples. A cell may have undergone 10, 20, 30, 40 or more doublings in vitro or ex vivo so that it is enriched compared to its original numbers in vivo or in its original tissue environment (e.g., bone marrow, peripheral blood, adipose tissue, etc.)
"MAPC" is an acronym for "multipotent adult progenitor cell." It refers to a non-embryonic stem cell that can give rise to cell lineages of all three germ layers (i.e., endoderm, mesoderm and ectoderm) upon differentiation. Like embryonic stem cells, human MAPCs express telomerase, Oct 3/4 (i.e., Oct 3A), rex-1, rox-1 and sox-2, and may express SSEA-4. The term "adult" in MAPC is non-restrictive. It refers to a non-embryonic somatic cell.
MAPCs constitutively express Oct 3/4 and high levels of telomerase (Jiang, Y. et al, Nature, 418:41 (2002); Exp Hematol, 30:896, 2002). MAPCs derived from human, mouse, rat or other mammals appear to be the only normal, non-malignant, somatic cell (i.e., non-germ cell) known to date to express very high levels of telomerase even in late passage cells. The telomeres are extended in MAPCs and they are karyotypically normal. Because MAPCs injected into a mammal can migrate to and assimilate within multiple organs, MAPCs are self-renewing stem cells.
"Multipotent," with respect to the term in "MAPC," refers to the ability to give rise to cell lineages of more than one primitive germ layer (i.e., endoderm, mesoderm and ectoderm) upon differentiation, such as all three. This term is not used consistently in the literature.
"Pluripotent" as used herein means any cell that, when exposed to Wnt3a and Activin A at the specified amounts, gives rise to cells with a definitive endodermal phenotype. Such cells may have the ability to give rise to cell lineages of more than one primitive germ layer (i.e., endoderm, mesoderm and ectoderm) upon differentiation, such as all three. "Primordial embryonic germ cells" (PG or EG cells) can be cultured and stimulated to produce many less differentiated cell types.
"Progenitor cells" are cells produced during differentiation of a stem cell that have some, but not all, of the characteristics of their terminally-differentiated progeny. The term "progenitor" as used in the acronym "MAPC" does not limit these cells to a particular lineage. A hepatocyte progenitor is any cell in the hepatocyte lineage that is less differentiated than a hepatocyte.
"Self-renewal" refers to the ability to produce replicate daughter stem cells having differentiation potential that is identical to those from which they arose. A similar term used in this context is "proliferation." "Stem cell" means a cell that can undergo self-renewal (i.e., progeny with the same differentiation potential) and also produce progeny cells that are more restricted in differentiation potential. Within the context of the invention, a stem cell would also encompass a more differentiated cell that has dedifferentiated, for example, by nuclear transfer, by fusions with a more primitive stem cell, by introduction of specific transcription factors, or by culture under specific conditions. See, for example, Wilmut et al, Nature, 385:810-813 (1997); Ying et al, Nature, 416:545-548 (2002); Guan et al, Nature, 440:1199- 1203 (2006); Takahashi et al, Cell, 126:663-676 (2006); Okita et al, Nature, 448:313-317 (2007); and Takahashi et al, Cell, 131 :861-872 (2007). Dedifferentiation may also be caused by the administration of certain compounds or exposure to a physical environment in vitro or in vivo that would cause the dedifferentiation. Stem cells also may be derived from abnormal tissue, such as a teratocarcinoma and some other sources such as embryoid bodies (although these can be considered embryonic stem cells in that they are derived from embryonic tissue, although not directly from the inner cell mass).
"Subject" means a vertebrate, such as a mammal, such as a human. Mammals include, but are not limited to, humans, dogs, cats, horses, cows and pigs.
The term "therapeutically effective amount" refers to the amount determined to produce any therapeutic response in a mammal. For example, effective amounts of the compounds, agents or drugs may prolong the survivability of the patient, and/or inhibit overt clinical symptoms. Treatments that are therapeutically effective within the meaning of the term as used herein, include treatments that improve a subject's quality of life even if they do not improve the disease outcome per se. Such therapeutically effective amounts are ascertained by one of ordinary skill in the art through routine application to subject populations such as in clinical and pre-clinical trials. Thus, to "treat" means to deliver such an amount.
"Treat," "treating" or "treatment" are used broadly in relation to the invention and each such term encompasses, among others, preventing, ameliorating, inhibiting, or curing a deficiency, dysfunction, disease, or other deleterious process, including those that interfere with and/or result from a therapy. As used herein, the terms "compound", "molecule" or "agent" are used interchangeably and broadly to refer to natural, synthetic or semi-synthetic molecules or compounds. The term: "molecule" therefore denotes for example chemicals, macromolecules, cell or tissue extracts (from plants or animals) and the like. Non-limiting examples of molecules include nucleic acid molecules including oligonucleotides (like antisense or aptamers) and small interfering RNAs, antigene therapeutics, peptides, antibodies, carbohydrates, small molecules, pharmaceutical agents, and/or cellular therapies. The agents can be selected and screened by a variety of means including random screening, rational selection and by rational design using for example protein or ligand modeling methods such as computer modeling. The terms "rationally selected" or "rationally designed" are meant to define compounds which have been chosen based on the configuration of interacting domains of the present invention. As will be understood by the person of ordinary skill, macromolecules having non-naturally occurring modifications are also within the scope of the term "molecule". For example, peptidomimetics, well known in the pharmaceutical industry and generally referred to as peptide analogs can be generated by modeling as mentioned above. Thus analogs or functional derivatives of the compounds of this invention, which can be made by several methods as is known by a person skilled in the art, are also contemplated within the scope of this invention. The molecules identified in accordance with the teachings of the present invention have a therapeutic value in diseases or conditions associated with hepatitis virus infection, more particularly, HCV infection. Alternatively, the molecules identified in accordance with the teachings of the present invention find utility in the development of more efficient anti- hepatitis virus compounds, more particularly, anti-HCV compounds.
As used herein, the designation "functional derivative" denotes, in the context of a functional derivative of a sequence whether a nucleic acid or amino acid sequence, a molecule that retains a biological activity (either functional or structural) that is substantially similar to that of the original sequence. Substantially similar means in terms of nucleic acid or amino acid sequence molecules, a molecule that has at least 50%, more preferably at least 70%>, yet more preferably 80%, still more preferably 90%, again more preferable 95% and most preferably at least 98% nucleic acid or amino acid sequence identity with said nucleic acid or amino acid sequence molecule. This functional derivative or equivalent may be a natural derivative or may be prepared synthetically. Such derivatives include amino acid sequences having substitutions, deletions, or additions of one or more amino acids, provided that the biological activity of the protein is conserved. The same applies to derivatives of nucleic acid sequences which can have substitutions, deletions, or additions of one or more nucleotides, provided that the biological activity of the sequence is generally maintained. When relating to a protein sequence, the substituting amino acid generally has chemico-physical properties which are similar to that of the substituted amino acid. The similar chemico-physical properties include, similarities in charge, bulkiness, hydrophobicity, hydrophylicity and the like. The term "functional derivatives" is intended to include fragments, segments, variants, analogs, homologues or chemical derivatives of the subject matter of the present invention. In another embodiment, the present invention relates to an assay to screen for drugs for the treatment and/or prevention of a hepatotropic infection, more particularly a HCV infection. In a particular embodiment, such assays can be designed using cells from patients infected with such a hepatotropic virus (eg. HCV) having a known genotype. In accordance with the present invention, there is also provided a method for identifying, from a library of compounds, a compound with therapeutic effect on a hepatotropic infection, more particularly a HCV infection comprising providing a screening assay comprising a measurable biological activity of such a hepatotropic viral protein or gene, such as a HCV protein or gene (e.g. "in vitro') or measuring infectivity, (viral release etc.), contacting the screening assay whether in vitro or "cellular" with a test compound; and detecting if the test compound modulates the biological activity of said protein or gene or the infectivity of the virus; wherein a test compound which modulates the biological activity or the infectivity is a compound with this therapeutic effect. The screening assay according to the present invention is preferably an "in vitro" screening assay, but it can also be performed "in vivo" such as for example in a suitable mouse model.
As used herein, "biological activity" refers to any detectable biological activity of hepatotropic viral gene or protein, such as a HCV gene or protein. This includes any physiological function attributable to said gene or protein.
In one embodiment, the invention provides assays for screening candidate or test compounds which interact with hepatotropic genes or proteins, such as HCV genes or proteins.
In one embodiment, an assay is a cell-based assay in which a cell activity producing a hepatotropic virus such as a HCV is contacted with a test compound and the ability of the test compound to modulate the infectivity of said hepatotropic virus at different steps in said hepatotropic virus complete life cycle, (e.g., attachment, entry into cells, replication, maturation etc).
The assays described above may be used as initial or primary screens to detect promising lead compounds for further development. Often, lead compounds will be further assessed in additional, different screens. Therefore, this invention also includes secondary anti- hepatotropic virus screens, such as anti-HCV screens, which may involve purified factors of said hepatotropic virus.
Tertiary screens may involve the study of the identified modulators in animal models for hepatotropic virus infection, such as animal models for HCV infection. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model. For example, a test compound identified as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, the toxicity of any test compound, including the test compound described in this invention, can be analyzed in the cells produced by the methods of this invention in the absence of any (hepatitis) viral infection.
The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries (peptide libraries) and small molecule libraries of compounds. Methods and Compositions of the Invention
The invention relates to a method for inducing stem cells to differentiate into hepatocyte cells or cells with a hepatocyte phenotype (i.e. in vitro culture system), which is suitable for the full replication cycle of hepatotropic pathogens; in particular hepatotropic viruses, more particularly hepatitis C virus (HCV), HAV, Dengue Fever Virus and Yellow Fever virus. The methods of the invention induce cells in culture to progress through the appropriate stages of hepatic development, thus recapitulating hepatic development in vitro and, as a result, give rise to cells having functional hepatic properties (e.g., biochemical and anatomical characteristics of hepatic cells), which support the complete replication of hepatotropic pathogens. The in vitro culture system of the present invention is not only useful for infection with viral hepatotropic pathogens, but also for infection with other non viral hepatotropic pathogens such as parasitic pathogens.
Culture methods of the invention comprise a sequential addition of hepatic differentiation factors (herein also referred to as growth factors) to cells, wherein there is:
(a) a first addition of about 5 ng/ml to about 500 ng/ml Wnt3a, more particularly about 50 ng/ml Wnt3a, and about 10 ng/ml to about 1,000 ng/ml ActivinA, more particularly about
100 ng/ml ActivinA; followed by an addition of Activin A without Wnt3A, wherein
Activin A is added at concentrations of about 10 ng/ml to about 1,000 ng/ml ActivinA, more particularly about 100 ng/ml ActivinA;
(b) a second addition of about 5 ng/ml to about 500 ng/ml BMP4, more particularly about 50 ng/ml BMP4; (c) a third addition of about 5 ng/ml to about 500 ng/ml FGF1, more particularly about 50 ng/ml FGF1;
(d) and a fourth addition of about 2 ng/ml to about 200 ng/ml HGF, more particularly about 20 ng/ml HGF.
In particular, at least in one, two or three of steps (a), (b), (c) and (d), only a single growth factor is used. More in particular, at least in one, two or three of steps (a), (b), and (c), only a single growth factor is used.
At each successive step, the culture is continued for at least four days. More particularly, the cells are cultured in the first step for about two days in ActicinA and Wnt3A containing medium and about two days in ActicinA containing medium; in the second step for about four days; in the third step for about four days; and in the fourth step for about six days. In one embodiment, cells are cultured with Wnt3a for about 2 days. At one or more steps, the cells are cultured in a medium containing a serum concentration from 0% to about 2%, more particularly about 0%. In one embodiment, cells are cultured in 0% serum throughout the complete differentiation protocol.
Additionally, at one or more steps, the cells are cultured in a medium containing about 10"4 M to about 10"7 M dexamethasone, more particularly about 10"6 M dexamethasone. Additionally, at one or more steps, the cells are cultured in a medium containing about 0.1 mg/ml to about 50 mg/ml insulin, more particularly about 2.5 mg/ml insulin.
Culture medium at each successive step of the methods of the present invention is prepared to contain only the growth factor(s) described above, and cells are washed between each step to reduce the presence of previously added growth factor(s). Alternatively, reduced concentrations of the previously provided factor(s) in a previous step can remain in the culture medium of the next step.
The methods of the present invention contemplate the use of any Wnt3a, Activin-A, BMP4, FGF1, and HGF known in the art and having conserved function, and from all species (e.g., orthologs from human, mouse, rat, monkey, pig and the like). The hepatic differentiation factors of the present invention are well known to those skilled in the art.
Suitable forms of Wnt3a, Activin-A, BMP4, FGF1, and HGF include, but are not limited to, isolated polypeptides, which are optionally recombinant, including whole proteins, partial proteins (e.g., domains) and peptide fragments. Fragments of a polypeptide preferably are those fragments that retain the distinct functional capability of the particular factor, which in the present invention generally relates to the ability to influence hepatic differentiation (the specific function of each factor is well known in the art). Such polypeptides also include, but are not limited to, fusion proteins and chimeric proteins. Short polypeptides can be synthesized chemically using well-established methods of peptide synthesis.
Cytokines may be replaced by small molecules that activate the same signal pathway, such as GSK3b inhibitor for Wnt3a; kinase activating molecules for the FGFs.
The culture methods of the present invention comprise a sequential addition of hepatic differentiation factors to cells. In the first step of the present invention, the hepatic differentiation factors Wnt3a and Activin A are added to the cells.
The concentration of Wnt3a that is added to the cells can range from about 5 ng/ml to about 500 ng/ml. However, the invention also encompasses sub-ranges of concentrations of Wnt3a. For example, from about 5-25 ng/ml, 25-50 ng/ml, 50-75 ng/ml, 75-100 ng/ml, 100-150 ng/ml, 150-300 ng/ml and 300-500 ng/ml. The preferred concentration of Wnt3a that is added to the cells is about 50 ng/ml. The duration of Wnt3a exposure used in the examples is two days. However, this may be changed to three, four, five or six days.
The concentration of Activin A that is added to the cells can range from about 10 ng/ml to about 1000 ng/ml. However, the invention also encompasses sub-ranges of concentrations of Activin A. For example, from about 10-25 ng/ml, 25-50 ng/ml, 50-75 ng/ml, 75-100 ng/ml, 100-125 ng/ml, 125-150 ng/ml, 150-175 ng/ml, 175-200 ng/ml, 200-400 ng/ml, 400-600 ng/ml, 600-800 ng/ml and 800-1000 ng/ml. The preferred concentration of Activin A that is added to the cells is about 100 ng/ml. The duration of Activin A exposure used in the examples is four days (two days in the presence of Wnt3 A followed by two more days in the absence of Wnt3A). However, this may be changed to five, six or seven days.
In the second step of the present invention, the hepatic differentiation factor BMP4 is added to the cells. The concentration of BMP4 that is added to the cells can range from about 5 ng/ml to about 500 ng/ml. However, the invention also encompasses sub-ranges of concentrations of BMP4. For example, from about 5-10 ng/ml, 10-20 ng/ml, 20-30 ng/ml, 30-40 ng/ml, 40-50 ng/ml, 50-60 ng/ml, 60-70 ng/ml, 70-80 ng/ml, 80-90 ng/ml, 90-100 ng/ml, 100-200 ng/ml, 200-300 ng/ml, 300-400 ng/ml and 400-500 ng/ml. The preferred concentration of BMP4 that is added to the cells is about 50 ng/ml. The duration of BMP4 exposure used in the examples is four days. However, this may be changed to five, six, or seven days.
In the third step of the present invention, the hepatic differentiation factor FGFl (also indicated as aFGF) is added to the cells.
The concentration of FGFl that is added to the cells can range from about 5 ng/ml to about 500 ng/ml. However, the invention also encompasses sub-ranges of concentrations of FGFl . For example, from about 5-10 ng/ml, 10-20 ng/ml, 20-30 ng/ml, 30-40 ng/ml, 40-50 ng/ml, 50-60 ng/ml, 60-70 ng/ml, 70-80 ng/ml, 80-90 ng/ml, 90-100 ng/ml, 100-200 ng/ml, 200-300 ng/ml, 300-400 ng/ml and 400-500 ng/ml. The preferred concentration of FGFl that is added to the cells is about 50 ng/ml. The duration of aFGF exposure used in the examples is four days. However, this may be changed to five, six, or seven days.
In the fourth step of the present invention, the hepatic differentiation factor HGF is added to the cells.
The concentration of HGF that is added to the cells can range from about 2 ng/ml to about 200 ng/ml. However, the invention also encompasses sub-ranges of concentrations of HGF. For example, from about 2-5 ng/ml, 5-10 ng/ml, 10-15 ng/ml, 15-20 ng/ml, 20-25 ng/ml, 25- 30 ng/ml, 30-35 ng/ml, 35-40 ng/ml, 40-50 ng/ml, 50-100 ng/ml, 100-150 ng/ml and 150-200 ng/ml. The preferred concentration of HGF that is added to the cells is about 20 ng/ml. The duration of HGF exposure used in the examples is 16 days. However, this may be changed to four, five, six, seven, eigth, nine, ten, eleven, twelf, thirteen, fourteen and fifteen days and can be as high as 30 days.
The present invention can be practiced, preferably, using stem cells of vertebrate species, such as humans, non-human primates, domestic animals, livestock, and other non-human mammals.
Stem cells have been identified in most tissues. The most well studied stem cell is the embryonic stem cell (ESC), as it has unlimited self-renewal and multipotent differentiation potential. These cells are derived from the inner cell mass of the blastocyst or can be derived from the primordial germ cells of a post-implantation embryo (embryonal germ cells or EG cells). ES and EG cells have been derived, first from mouse, and later, from many different animals, and more recently, from non-human primates and humans. When introduced into mouse blastocysts or blastocysts of other animals, ESCs can contribute to all tissues of the animal. ES and EG cells can be identified by positive staining with antibodies against SSEA1 (mouse) and SSEA4 (human). See, for example, U.S. Patent Nos. 5,453,357; 5,656,479; 5,670,372; 5,843,780; 5,874,301; 5,914,268; 6,110,739 6,190,910; 6,200,806; 6,432,711; 6,436,701, 6,500,668; 6,703,279; 6,875,607; 7,029,913; 7,112,437; 7,145,057; 7,153,684; and 7,294,508, each of which is incorporated by reference herein for teaching ESCs and methods of making and expanding ESCs. Accordingly, ESCs and methods for isolating and expanding ESCs are well-known in the art. Meanwhile a number of established embryonic stem cell lines have been developed in the art, and in a particular embodiment of the present invention, such established ES cell lines are used as ESC.
A number of transcription factors and exogenous cytokines have been identified that influence the potency status of embryonic stem cells in vivo. The first transcription factor to be described that is involved in stem cell pluripotency is Oct4. Oct4 belongs to the POU (Pit- Oct-Unc) family of transcription factors and is a DNA binding protein that is able to activate the transcription of genes, containing an octameric sequence called "the octamer motif within the promotor or enhancer region. Oct4 is expressed at the moment of the cleavage stage of the fertilized zygote until the egg cylinder is formed. The function of Oct3/4 is to repress differentiation inducing genes (i.e., FoxaD3, hCG) and to activate genes promoting pluripotency (FGF4, Utfl, Rexl). Sox2, a member of the high mobility group (HMG) box transcription factors, cooperates with Oct4 to activate transcription of genes expressed in the inner cell mass. It is essential that Oct3/4 expression in embryonic stem cells is maintained between certain levels. Overexpression or downregulation of >50% of Oct4 expression level will alter embryonic stem cell fate, with the formation of primitive endoderm/mesoderm or trophectoderm, respectively. In vivo, Oct4 deficient embryos develop to the blastocyst stage, but the inner cell mass cells are not pluripotent. Instead they differentiate along the extraembryonic trophoblast lineage. Sall4, a mammalian Spalt transcription factor, is an upstream regulator of Oct4, and is therefore important to maintain appropriate levels of Oct4 during early phases of embryology. When Sall4 levels fall below a certain threshold, trophectodermal cells will expand ectopically into the inner cell mass. Another transcription factor required for pluripotency is Nanog, named after a Celtic tribe "Tir Nan Og": the land of the ever young. In vivo, Nanog is expressed from the stage of the compacted morula, is subsequently defined to the inner cell mass and is downregulated by the implantation stage. Downregulation of Nanog may be important to avoid an uncontrolled expansion of pluripotent cells and to allow multilineage differentiation during gastrulation. Nanog null embryos, isolated at day 5.5, consist of a disorganized blastocyst, mainly containing extraembryonic endoderm and no discernable epiblast. Non-Embryonic
An example of a non-embryonic stem cell is adipose-derived adult stem cells (ADSCs) which have been isolated from fat, typically by liposuction followed by release of the ADSCs using collagenase. ADSCs are similar in many ways to MSCs derived from bone marrow, except that it is possible to isolate many more cells from fat. These cells have been reported to differentiate into bone, fat, muscle, cartilage and neurons. A method of isolation has been described in U.S. 2005/0153442.
Other non-embryonic cells reported to be capable of differentiating into cell types of more than one embryonic germ layer include, but are not limited to, cells from umbilical cord blood (see U.S. Publication No. 2002/0164794), placenta (see U.S. Publication No. 2003/0181269; umbilical cord matrix (Mitchell, K.E. et al, Stem Cells, 21 :50-60, 2003), small embryonic-like stem cells (Kucia, M. et al, J Physiol Pharmacol, 57 Suppl 5:5-18, 2006), amniotic fluid stem cells (Atala, A., J Tissue Regen Med, 1 :83-96, 2007), skin-derived precursors (Toma et al, Nat Cell Biol, 3:778-784, 2001), and bone marrow (see U.S. Publication Nos. 2003/0059414 and 2006/0147246), each of which is incorporated by reference herein for teaching these cells. Other stem cells that are known in the art include gastrointestinal stem cells, epidermal stem cells, and hepatic stem cells, which also have been termed "oval cells" (Potten, C, et al, Trans R Soc Lond B Biol Sci, 353:821-830 (1998);, Watt, F., Trans R Soc Lond B Biol Sci, 353:831 (1997); Alison et al, Hepatology, 29:678-683 (1998).
Strategies of Reprogramming Somatic Cells Several different strategies such as nuclear transplantation, cellular fusion, and culture induced reprogramming have been employed to induce the conversion of differentiated cells into an embryonic state. Nuclear transfer involves the injection of a somatic nucleus into an enucleated oocyte, which, upon transfer into a surrogate mother, can give rise to a clone ("reproductive cloning"), or, upon explantation in culture, can give rise to genetically matched embryonic stem (ES) cells ("somatic cell nuclear transfer," SCNT). Cell fusion of somatic cells with ES cells results in the generation of hybrids that show all features of pluripotent ES cells. Explantation of somatic cells in culture selects for immortal cell lines that may be pluripotent or multipotent. At present, spermatogonial stem cells are the only source of pluripotent cells that can be derived from postnatal animals. Transduction of somatic cells with defined factors can initiate reprogramming to a pluripotent state. These experimental approaches have been extensively reviewed (Hochedlinger and Jaenisch, Nature, 441 : 1061-1067 (2006) and Yamanaka, S., Cell Stem Cell, 1 :39-49 (2007)).
Nuclear transplantation (NT), also referred to as somatic cell nuclear transfer (SCNT), denotes the introduction of a nucleus from a donor somatic cell into an enucleated ogocyte to generate a cloned animal such as Dolly the sheep (Wilmut et al, Nature, 385:810-813 (1997). The generation of live animals by NT demonstrated that the epigenetic state of somatic cells, including that of terminally differentiated cells, while stable, is not irreversible fixed but can be reprogrammed to an embryonic state that is capable of directing development of a new organism. In addition to providing an exciting experimental approach for elucidating the basic epigenetic mechanisms involved in embryonic development and disease, nuclear cloning technology is of potential interest for patient-specific transplantation medicine.
Fusion of Somatic Cells and Embryonic Stem Cells
Epigenetic reprogramming of somatic nuclei to an undifferentiated state has been demonstrated in murine hybrids produced by fusion of embryonic cells with somatic cells. Hybrids between various somatic cells and embryonic carcinoma cells (Solter, D., Nat Rev Genet, 7:319-327 (2006), embryonic germ (EG), or ES cells (Zwaka and Thomson, Development, 132:227-233 (2005)) share many features with the parental embryonic cells, indicating that the pluripotent phenotype is dominant in such fusion products. As with mouse (Tada et al., Curr Biol, 11 :1553-1558 (2001)), human ES cells have the potential to reprogram somatic nuclei after fusion (Cowan et al, Science, 309: 1369-1373(2005)); Yu et al, Science, 318:1917-1920 (2006)). Activation of silent pluripotency markers such as Oct4 or reactivation of the inactive somatic X chromosome provided molecular evidence for reprogramming of the somatic genome in the hybrid cells. It has been suggested that DNA replication is essential for the activation of pluripotency markers, which is first observed 2 days after fusion (Do and Scholer, Stem Cells, 22:941-949 (2004)), and that forced overexpression of Nanog in ES cells promotes pluripotency when fused with neural stem cells (Silva et al, Nature, 441 :997-1001 (2006)).
Culture-Induced Reprogramming Pluripotent cells have been derived from embryonic sources such as blastomeres and the inner cell mass (ICM) of the blastocyst (ES cells), the epiblast (EpiSC cells), primordial germ cells (EG cells), and postnatal spermatogonial stem cells ("maGSCsm" "ES-like" cells). The following pluripotent cells, along with their donor cell/tissue is as follows: parthogenetic ES cells are derived from murine oocytes (Narasimha et al, Curr Biol, 7:881-884 (1997)); embryonic stem cells have been derived from blastomeres (Wakayama et al., Stem Cells, 25:986-993 (2007)); inner cell mass cells (source not applicable) (Eggan et al, Nature, 428:44-49 (2004)); embryonic germ and embryonal carcinoma cells have been derived from primordial germ cells (Matsui et al, Cell, 70:841-847 (1992)); GMCS, maSSC, and MASC have been derived from spermatogonial stem cells (Guan et al, Nature, 440:1199-1203 (2006); Kanatsu-Shinohara et al, Cell, 119: 1001-1012 (2004); and Seandel et al, Nature, 449:346-350 (2007)); EpiSC cells are derived from epiblasts (Brons et al, Nature, 448:191- 195 (2007); Tesar et al, Nature, 448: 196-199(2007)); parthogenetic ES cells have been derived from human oocytes (Cibelli et al, Science, 295L819 (2002); Revazova et al, Cloning Stem Cells, 9:432-449 (2007)); human ES cells have been derived from human blastocysts (Thomson et al, Science, 282: 1145-1147 (1998)); MAPC have been derived from bone marrow (Jiang et al, Nature, 418:41-49 (2002); Phinney and Prockop, Stem Cells, 25:2896-2902 (2007)); cord blood cells (derived from cord blood) (van de Ven et al, Exp Hematol, 35: 1753-1765 (2007)); neurosphere derived cells derived from neural cell (Clarke et al, Science, 288: 1660-1663 (2000)). Donor cells from the germ cell lineage such as PGCs or spermatogonial stem cells are known to be unipotent in vivo, but it has been shown that pluripotent ES-like cells (Kanatsu-Shinohara et al, Cell, 119: 1001-1012 (2004) or maGSCs (Guan et al, Nature, 440: 1199-1203 (2006), can be isolated after prolonged in vitro culture. While most of these pluripotent cell types were capable of in vitro differentiation and teratoma formation, only ES, EG, EC, and the spermatogonial stem cell-derived maGCSs or ES-like cells were pluripotent by more stringent criteria, as they were able to form postnatal chimeras and contribute to the germline. Recently, multipotent adult spermatogonial stem cells (MASCs) were derived from testicular spermatogonial stem cells of adult mice, and these cells had an expression profile different from that of ES cells (Seandel et al., Nature, 449:346-350 (2007)) but similar to EpiSC cells, which were derived from the epiblast of postimplantation mouse embryos (Brons et al, Nature, 448:191-195 (2007); Tesar et al, Nature, 448: 196-199 (2007)).
Reprogramming by Defined Transcription Factors
Takahashi and Yamanaka have reported reprogramming somatic cells back to an ES-like state (Takahashi and Yamanaka, Cell, 126:663-676 (2006)). They successfully reprogrammed mouse embryonic fibroblasts (MEFs) and adult fibroblasts to pluripotent ES- like cells after viral-mediated transduction of the four transcription factors Oct4, Sox2, c- myc, and Klf4 followed by selection for activation of the Oct4 target gene Fbxl5 (Figure 2A). Cells that had activated Fbxl5 were coined iPS (induced pluripotent stem) cells and were shown to be pluripotent by their ability to form teratomas, although the were unable to generate live chimeras. This pluripotent state was dependent on the continuous viral expression of the transduced Oct4 and Sox2 genes, whereas the endogenous Oct4 and Nanog genes were either not expressed or were expressed at a lower level than in ES cells, and their respective promoters were found to be largely methylated. This is consistent with the conclusion that the Fbxl5-iPS cells did not correspond to ES cells but may have represented an incomplete state of reprogramming. While genetic experiments had established that Oct4 and Sox2 are essential for pluripotency (Chambers and Smith, Oncogene, 23:7150-7160 (2004); Ivanona et al, Nature, 442:5330538 (2006); Masui et al, Nat Cell Biol, 9:625-635 (2007)), the role of the two oncogenes c-myc and Klf4 in reprogramming is less clear. Some of these oncogenes may, in fact, be dispensable for reprogramming, as both mouse and human iPS cells have been obtained in the absence of c-myc transduction, although with low efficiency (Nakagawa et al, Nat Biotechnol, 26:191-106 (2008); Werning et al, Nature, 448:318-324 (2008); Yu et al, Science, 318: 1917-1920 (2007)).
MAPC is an acronym for "multipotent adult progenitor cell" (non-ES, non-EG, non-germ). Genes found in ES cells also have been found in MAPCs (e.g., telomerase, Oct 3/4, rex-1, rox-1, sox-2). Oct 3/4 (Oct 3 A in humans) appears to be specific for ES and germ cells. MAPC represents a more primitive progenitor cell population than MSC and demonstrates differentiation capability encompassing the epithelial, endothelial, neural, myogenic, hematopoietic, osteogenic, hepatogenic, chondrogenic and adipogenic lineages (Verfaillie, CM., Trends Cell Biol, 12:502-8, 2002, Jahagirdar, B.N., et al, Exp Hematol, 29:543-56, 2001; Reyes, M. and CM. Verfaillie, Ann N Y Acad Sci, 938:231-233, 2001; Jiang, Y. et al, Exp Hematol, 30896-904, 2002; and Jiang, Y. et al, Nature, 418:41-9, 2002).
Human MAPCs are described in U.S. Patent 7,015,037 and U.S. Application No. 10/467,963. MAPCs have been identified in other mammals. Murine MAPCs, for example, also are described in U.S. Patent 7,015,037 and U.S. Application No. 10/467,963. Rat MAPCs also are described in U.S. Application No. 10/467,963.
In general, cells useful for the invention can be maintained and expanded in culture medium that is available to and well-known in the art. Such media include, but are not limited to, Dulbecco's Modified Eagle's Medium® (DMEM), DMEM F12 medium®, Eagle's Minimum Essential Medium®, F-12K medium®, Iscove's Modified Dulbecco's Medium® and RPMI-1640 medium®. Many media are also available as low-glucose formulations, with or without sodium pyruvate.
Also contemplated in the present invention is supplementation of cell culture medium with mammalian sera. In certain embodiments of the present invention, such mammalian sera concentrations range between 0% and 2%, more particularly the serum concentration is 0%. Sera often contain cellular factors and components that are necessary for viability and expansion. Examples of sera include fetal bovine serum (FBS), bovine serum (BS), calf serum (CS), fetal calf serum (FCS), newborn calf serum (NCS), goat serum (GS), horse serum (HS), human serum, chicken serum, porcine serum, sheep serum, rabbit serum, serum replacements and bovine embryonic fluid. It is understood that sera can be heat-inactivated at 55-65°C if deemed necessary to inactivate components of the complement cascade.
Additional supplements also can be used advantageously to supply the cells with the necessary trace elements for optimal growth and expansion. Such supplements include insulin, transferrin, sodium selenium and combinations thereof. These components can be included in a salt solution such as, but not limited to, Hanks' Balanced Salt Solution® (HBSS), Earle's Salt Solution®, antioxidant supplements, MCDB-201® supplements, phosphate buffered saline (PBS), ascorbic acid and ascorbic acid-2-phosphate, as well as additional amino acids. Many cell culture media already contain amino acids, however, some require supplementation prior to culturing cells. Such amino acids include, but are not limited to, L-alanine, L-arginine, L-aspartic acid, L-asparagine, L-cysteine, L-cystine, L- glutamic acid, L-glutamine, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L- methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine. It is well within the skill of one in the art to determine the proper concentrations of these supplements. Hormones also can be advantageously used in the cell cultures of the present invention and include, but are not limited to, D-aldosterone, diethylstilbestrol (DES), dexamethasone, β- estradiol, hydrocortisone, insulin, prolactin, progesterone, somatostatin/human growth hormone (HGH), thyrotropin, thyroxine and L-thyronine.
Lipids and lipid carriers also can be used to supplement cell culture media, depending on the type of cell and the fate of the differentiated cell. Such lipids and carriers can include, but are not limited to, cyclodextrin (α, β, γ), cholesterol, linoleic acid conjugated to albumin, linoleic acid and oleic acid conjugated to albumin, unconjugated linoleic acid, linoleic-oleic- arachidonic acid conjugated to albumin and oleic acid unconjugated and conjugated to albumin, among others. Also contemplated in the present invention is the use of feeder cell layers. Feeder cells are used to support the growth of fastidious cultured cells such as ES cells. Feeder cells are normal cells that have been inactivated by γ-irradiation. In culture, the feeder layer serves as a basal layer for other cells and supplies cellular factors without further growth or division of their own (Lim, J.W. and Bodnar, A., 2002). Examples of feeder layer cells are typically human diploid lung cells, mouse embryonic fibroblasts and Swiss mouse embryonic fibroblasts, but can be any post-mitotic cell that is capable of supplying cellular components and factors that are advantageous in allowing optimal growth, viability and expansion of stem cells. In many cases, feeder cell layers are not necessary to keep ES cells in an undifferentiated, proliferative state, as leukemia inhibitory factor (LIF) has anti- differentiation properties. Therefore, supplementation with LIF can be used to maintain MAPC in some species in an undifferentiated state.
Cells may be cultured in low-serum or serum-free culture medium. Serum-free medium used to culture MAPCs is described in U.S. Patent 7,015,037. Many cells have been grown in serum-free or low-serum medium. In this case, the medium is supplemented with one or more growth factors. Commonly used growth factors include, but are not limited to, bone morphogenic protein, basis fibroblast growth factor, platelet-derived growth factor and epidermal growth factor. See, for example, U.S. Patent Nos. 7,169,610; 7,109,032; 7,037,721; 6,617,161; 6,617,159; 6,372,210;6,224,860; 6,037,174; 5,908,782; 5,766,951; 5,397,706; and 4,657,866; all incorporated by reference herein for teaching growing cells in serum-free medium.
Cells in culture can be maintained either in suspension or attached to a solid support, such as extracellular matrix components. Stem cells often require additional factors that encourage their attachment to a solid support, such as type I and type II collagen, chondroitin sulfate, fibronectin, "superfibronectin" and fibronectin-like polymers, gelatin, poly-D and poly-L- lysine, thrombospondin and vitronectin. One embodiment of the present invention utilizes fibronectin. See, for example, Ohashi et al, Nature Medicine, 13:880-885 (2007); Matsumoto et al, J Bioscience and Bioengineering, 105:350-354 (2008); Kirouac et al, Cell Stem Cell, 3:369-381 (2008); Chua et al, Biomaterials, 26:2537-2547 (2005); Drobinskaya et al, Stem Cells, 26:2245-2256 (2008); Dvir-Ginzberg et al, FASEB J, 22: 1440-1449 (2008); Turner et al, J Biomed Mater Res Part B: Appl Biomater, 82B: 156-168 (2007); and Miyazawa et al, Journal of Gastroenterology and Hepatology, 22: 1959-1964 (2007)).
Cells may also be grown in "3D" (aggregated) cultures. An example is U.S. Provisional Patent Application No. 61/022,121, filed January 18, 2008.
Once established in culture, cells can be used fresh or frozen and stored as frozen stocks, using, for example, DMEM with 40% FCS and 10% DMSO. Other methods for preparing frozen stocks for cultured cells also are available to those skilled in the art.
Methods of identifying and subsequently separating differentiated cells from their undifferentiated counterparts can be carried out by methods well known in the art. Cells that have been induced to differentiate using methods of the present invention can be identified by selectively culturing cells under conditions whereby differentiated cells outnumber undifferentiated cells. Similarly, differentiated cells can be identified by morphological changes and characteristics that are not present on their undifferentiated counterparts, such as cell size and the complexity of intracellular organelle distribution. Also contemplated are methods of identifying differentiated cells by their expression of specific cell-surface markers such as cellular receptors and transmembrane proteins. Monoclonal antibodies against these cell-surface markers can be used to identify differentiated cells. Detection of these cells can be achieved through fluorescence activated cell sorting (FACS) and enzyme-linked immunosorbent assay (ELISA). From the standpoint of transcriptional upregulation of specific genes, differentiated cells often display levels of gene expression that are different from undifferentiated cells. Reverse-transcription polymerase chain reaction, or RT-PCR, also can be used to monitor changes in gene expression in response to differentiation. Whole genome analysis using microarray technology also can be used to identify differentiated cells.
Accordingly, once differentiated cells are identified, they can be separated from their undifferentiated counterparts, if necessary. The methods of identification detailed above also provide methods of separation, such as FACS, preferential cell culture methods, ELISA, magnetic beads and combinations thereof. One embodiment of the present invention comtemplates the use of FACS to identify and separate cells based on cell-surface antigen expression.
Uses for the Cells
The liver plays a major role in metabolism, including plasma protein synthesis (e.g. albumin and coagulation factors), glycogen storage, decomposition of red blood cells, and detoxification. One of the major impediments to the use of hepatocytes in at least one area of medicine is, in general, the scarcity of human hepatocytes that are available, eg. for developing a screening system of hepatotropic virus infectable (and replication supportable) cells which can be used to screen for antiviral drugs, more in particular hepatitis drugs and more particularly inhibitors of HCV. In another example, such cells (hepatocytes) can be used to screen for drugs for other (non-viral) hepatotropic pathogens as well.
Study of and drug development against human hepatitis viruses
A huge area where generating hepatocytes from stem cells, eg. as described in the present invention, would aid human health significantly is in the area of viral hepatitis . Most in vitro studies on the hepatitis viruses have involved primary human hepatocytes. However, again, the limited availability of human tissue and also subject to the variability of the source material impedes these types of studies.
Accordingly, the cells of the invention can be used in such studies and in drug development.
In the present invention, cell culture conditions were developed in view of (marker)gene expression and optimized from conditions that were previously developed (Roelandt, P., et al. 2010, and WO2010049752), for a detailed description, see Example 1 and Figure 3 in this specification. The inventors developed specific cell culture conditions to successfully produce cells that express phenotypes of hepatocytes. Next to the use in the hepatitis field, such (hepatocyte) differentiated cells are useful, amongst others, in the field of therapy of liver failure and for pharmaceutical testing.
Therapy of liver failure with hepatocyte transplantation:
One of the unique features of the liver is its enormous natural regeneration ability. This regeneration is due in large part to the re-entry of terminally differentiated hepatocytes in the cell cycle, resulting in multiple cell divisions to regenerate the liver. When the hepatocytes are damaged, liver stem/progenitor cells, termed oval cells in rodents, located in the periportal zone, are activated and differentiate to mature hepatocytes. Hence, most liver diseases ending in liver failure are caused by a combination of decreased proliferation of hepatocytes and exhaustion of the stem/progenitor cell pool. Liver failure is caused by a number of disorders, including cirrhosis due to infections, excessive alcohol consumption, genetic and idiopatic reasons. In addition, acute liver failure is caused by ingestion of certain drugs or foods. Liver transplantation is the only successful treatment for end stage liver disease, and is in many instances also the only curative therapy for certain forms of genetic disorders of the liver. Many liver disorders treated by whole liver transplantation result from hepatocyte dysfunction. As a consequence, there has been great interest in hepatocyte transplantation for the treatment of acute and chronic liver failure, as well as inherited metabolic disorders. There is significant evidence that grafted hepatocytes can assume the full range of liver functions in vivo. Hepatocyte transplantation has several advantages over whole liver transplant: lower morbidity, a single donor organ can be used for several recipients, cells can be cryopreserved, and cells grafts are less immunogenic than whole organ grafts. However, lack of donor cells curtails further exploration of this therapy.
Accordingly the invention is also directed to methods of treating liver deficiencies by administering the (differentiated) cells of the invention to a subject with the liver deficiency. Such deficiencies include, but are not limited to, toxic liver disease, metabolic liver disease, acute liver necrosis, effects of acetaminophen, hemochromatosis, Wilson's Disease, Crigler Najar, hereditary tyrosinemia, familial intrahepatic cholestatis type 3, ornithine transcarbamylase (OTC) deficiency, and urea cycle disorder.
Further diseases include, but are not limited to viral hepatitis, chronic viral hepatitis A, B, C, acute hepatitis A, B, C, D, E, cytomegalovirus and herpes simplex virus; liver dysfunction in other infectious diseases such as, without limitation, toxoplasmosis, hepatosplenic schistosomiasis, liver disease in syphilis, leptospirosis and amoebiasis; metabolic diseases such as, without limitation, haemochromatosis, Gilbert's syndrome, Dubin- Johnson syndrome and Rotor's syndrome; alcoholic liver disease such as, without limitation, fatty liver, fibrosis, sclerosis and cirrhosis; and toxic liver disease. Bioartificial liver (BAL) devices
In patients with terminal liver failure, the use of bioartificial liver devices has been proposed to bridge the time to liver transplantation. BAL devices are designed to support the detoxification functions performed by the liver, hence decreasing the risk and severity of CNS complications associated with acute liver failure. BAL devices could benefit three groups of patients; those with fulminant hepatic failure, those waiting for an imminent transplant, and those with early failure of a liver transplant. Although some positive results have been seen in patients with liver failure, further exploration of the usefulness of BAL devices has been hampered by lack of suitable cells. Currently, tumor-derived cell lines or animal cells, which might be associated with possible tumor cell seeding, immune responses, and xeno-zoonoses, are used. The availability of cells with full mature hepatic function of human origin, would enable investigators to further test and optimize BAL devices to bridge patients till the liver spontaneously regenerates or a donor-liver is available. Although clinical trials have in general not been successful, some encouraging results have been seen in patients with acute liver failure. Accordingly, the (differentiated) cells of the invention can be used in such bioartificial liver devices.
Pharmaceutical testing is moving more and more from in vivo experimentation to in vitro studies. Over the past decade, in vitro models were established, such as precision-cut liver slices, primary hepatocytes, and liver cell lines. A few studies examining the relevance of drug testing with hepatocyte cell lines, found that cell lines poorly reproduce and predict drug metabolism and hepatotoxicity as opposed to primary hepatocytes or liver slices. Thus, primary human hepatocytes are the "gold standard" for in vitro drug testing. However, the limited supply of human hepatocytes and the fact that such hepatocytes may not represent the genetic variation in society, limit the possibility of detecting potential drug toxicities. Consequently, development of stem cells from a diverse group of donors (for instance by generating IPS cells) and differentiation to hepatocytes with differing cytochrome P450 profiles would allow drug testing to more closely examine and predict potential problems for particular groups or individuals. Accordingly, the (differentiated) cells of the invention can be used in such testing methods. The present invention is additionally described by way of the following illustrative, non- limiting example that provides a better understanding of the present invention and of its many advantages.
EXAMPLES EXAMPLE 1: Hepatitis C Virus infection
1. Cells, Virus and Compounds
Human embryonic stem cells (hESC-H9 and hESC-Hl, WiCell, Madison, WI) and iPSC (derived by co-transduction with OCT4, SOX2, KLF4 and cMYC in the fibroblast cell line BJ1) were used. The iPSC line was characterized by embryoid body and teratoma formation, and could undergo directed differentiation to hepatocyte- and neuron-like cells as determined by RT-qPCR and immunostaining. The transgenes were silenced, as determined by RT- qPCR.
HCV JFH cell-culture mutant virus, described by Delgrange et a/.2007, was used with a specific-infectivity titer of 1 :400 (ratio between FFU/mL:HCV RNA copies number Zhong, J., et al. 2006). hSC were differentiated towards liver-specific progeny using a modified and optimized differentiation protocol, as compared to the initial protocol described earlier (Roelandt, P., et al. 2010, and WO2010049752) (Figure 3). In brief, the optimized protocol used in this studay: ActA (100ng/ml)-Wnt3A (50ng/ml) day 0-2, ActA (lOOng/ml) day 2-4, BMP4 (50ng/ml) day 4-8, FGF1 (50ng/ml) day 8-12, HGF (20ng/ml) day 12-28, and no serum was added during the complete optimized protocol. In parallel to stem cell cultures, the highly permissive Huh7.5.1 cells were used as controls.
At the time of infection (day 20 of differentiation), the monolayer of cells in 12-well cell culture plates were inoculated with the HCV virus stock for 48h at 37°C. Following two to three washes, fresh medium was added for additional 72h. For the antiviral assays, 10 μΜ of the non-nucleoside HCV polymerase inhbitior HCV-796 or 1 μΜ of the HCV NS3 protease inhibitor VX-950 was added to the cultures (n=2). HCV NS3 protease inhibitor VX-950 and the non-nucleoside NS5B polymerase inhibitor (benzofuran) HCV-796 were synthesized as described before (references in Paeshuyse J, et al, 2008). Supernatants were collected every 3 days. 2. Immunocytochemistry
hSC progeny was fixed using 4% Neutral Buffered Formalin (NBF) for 15 minutes at room temperature. Permeabilization and blocking was done for 15 minutes using phosphate buffered saline (PBS) containing 0.2% Triton X-100 (Acros Organics) and 3% donkey serum (Jackson). The cells were then incubated with the mixture of primary antibodies diluted in PBS overnight at 4°C. After three washes with PBS, the cells were incubated with the mixture of secondary antibodies and Hoechst (Sigma) for 30 minutes at room temperature. Primary antibodies and dilutions: mouse anti-CD81 (BD Biosciences 555675, 1 : 100), rabbit anti-SR-Bl (Abeam AB3, 1 : 1000), mouse anti-HNF4a (Abeam AB41898, 1 :200), rabbit anti-ALB (Dako A0001, 1 :4000), mouse anti-CYP3A4/5 (BD Biosciences WB-MAB-3A, 1 :250) and rabbit anti-HCV NS5A (a kind gift of Prof. R. Bartenschlager, 1 :2000). Isotypes: mouse IgGi (BD Biosciences 550878), mouse IgG2A (Sigma M9144), rabbit serum (Dako X0902). Secondary antibodies (Invitrogen, 1 :500): goat anti-mouse Alexa 488, chicken anti- rabbit Alexa 488, goat anti-mouse Alexa 555, goat anti-rabbit Alexa 555. Quantification of cells was performed using Zeiss Axio Vision Software version 4.8.1 on >5 randomly taken pictures.
3. RNA extraction and RT-qPCR quantification
For RNA isolation from uninfected hSC-progeny, the RNeasy Mini-kit/Micro-kit (Qiagen) was used. DNase treatment was performed using Turbo DNase kit (Ambion). cDNA synthesis was performed from 1 μg of RNA with Superscript III First-Strand synthesis system (Invitrogen). RT-qPCR was performed with SYBR Green Platinum SYBR green qPCR Supermix-UDG (Invitrogen) in an Eppendorf Realplex/ABI 7000 (Eppendorf) equipment. RT-qPCR primers: CD81 (forward 5'-ATG TGA AGC AGT TCT ATG ACC-3', reverse 5'-TCA TCT CGA AGA TCA TGA TCA C-3'), CLDNl (forward 5'-GCG CGA TAT TTC TTC TTG CAG-3', reverse 5'-GCA GGT TTT GGA TAG GGC CT-3'), CLDN6 (forward 5'-AGA AGG ATT CCA AGG CCC G-3', reverse 5'-GAT GTT GAG TAG CGG GCC AT-3'), LDL-R (forward 5'-CAA GGA CAA ATC TGA CGA GG-3', reverse 5'-AGA GTG TCA CAT TAA CGC AG-3') and SR-B1 (forward 5'-TGA ACT GCT CTG TGA AAC TG-3', reverse 5'-AAT AGC ATT TCT CTT GGC TCC-3').
RNA was extracted from supernatants of infected hSC-progeny using QIAamp Viral RNA kit (Qiagen) according to manufacturer's instructions. Intracellular RNA from infected hSC- progeny was extracted using the RNAeasy kit (Qiagen). A 25 μΐ RT-qPCR contains 6.25 μΐ of 2x reaction buffer (Eurogentec), 12.65 μΐ H20, 5 μΐ total cellular R A extract and 200 nmol/L SF-JFH86 (5'-TGG CGT TAG TAT GAG TGT CGT AC A GCC TCC A-3'), and 200 nmol/L SR-JFH194 (5 '-AAA GGA CCC AGT CTT CCC GGC AAT T-3'), and 6 pmol/L prob (5'-FAM-TGG TCT GCG GAA CCG GTG AGT ACA CC-TAMRA-3').
4. Transmission electron microscopy
hSC-progeny was washed twice with PBS and scraped to obtain cell clusters. The fragments were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate. Following post- fixation in 1% osmium tetroxide and 0.1 mol/L of phosphate buffer, the samples were dehydrated in graded series of alcohol and embedded in epoxy resin. Ultrathin sections were cut, stained with uranyl acetate and lead citrate, and examined using a Zeiss EM 900 electron microscope (Oberkochen, Germany).
5. Quantification of albumin, HCV core protein and ApoBlOO in supernatans
Albumin was measured using a quantitative ELISA kit (Bethyl) as per manufacturer's protocol. HCV core protein and ApoBlOO were measured using Ortho® HCV core antigen ELISA (Wako Chemicals) and Alerchek's Human ApoBlOO ELISA, respectively, as previously described (Nahmias, Y., et al. 2008). 6. Secondary infection immunofluorescence assay
The collected supernatants were used to inoculate na'ive Huh7.5.1 in 8 well chamber slides (100 μΐ in 300 μΐ complete DMEM medium). After 72 hours, the medium was removed and cells were washed twice with PBS, fixed for 20 minutes with 3.5%> paraformaldehyde and permeabilized with 0.5% Triton X-100 for 15 minutes. Cells were washed with PBS then blocked for 20 minutes at room temperature with IT-Image signal enhancer (Invitrogen) and incubated with rabbit polyclonal anti-HCV NS5A antibody (1 :2000) diluted in 5% normal goat serum for 60 minutes, followed by goat anti-rabbit Alexa 488 (Invitrogen) for 45 minutes in dark. Finally, cells were washed for 10 minutes in PBS and maintained in prolong antifade mounting medium with DAPI (Invitrogen). 7. Statistics
Comparison between different conditions was analyzed using a two-tailed Student's t-test. P- values < 0.05 were considered significant, n indicates number of experiments.
8. Ethics All studies using human ESC and the generation of the hiPSC line were approved by the ethical committee for the use of human subjects in research, and teratoma studies with hiPSC in immunodeficient mice by the ethical committee for use of animals in research of Catholic University of Leuven, Belgium.
II. Results In this example, we demonstrate that hepatocytes derived from human embryonic stem cells (hESC) (H9 and HI cells) or hiPSC can support the complete HCV replication cycle including the production of infectious virus.
We first studied by means of RT-qPCR and immunocytochemistry the expression on human stem cell (hSC) progeny of receptors crucial for the entry of HCV, namely CD81, claudin-1, claudin-6, LDL-receptor and scavenger receptor Bl (Figure 1A). All genes were expressed in day 20 progeny at levels between 5.2 and 11.6% of gene expression in primary hepatocytes. Immunocytochemistry confirmed the presence of SR-B1 and CD 81 in respectively 40.1 + 16.7% (average ± standard deviation) of HNF4a+ and 46.0 ± 21.5% of ALB+ hepatoblast/ hepatocyte hSC progeny (Data not shown).
Following inoculation of hSC-progeny with HCV, cytoplasmic localization of NS5A could be demonstrated in 14.7 ± 12.7% of ALB+ and CYP3A4/5+ hepatocytes, indicating that HCV entry as well as transcription of the original viral RNA by the host cell ribosomes was successful (Data not shown). Nuclear localization of NS5A was also noted in 14.2 + 18.6% of ALB+ and CYP3A4/5+ hepatocytes, suggestive of nuclear translocation of NS5A.
Using qPCR on lysed cells and culture supernatants, viral RNA was detected for as long as 10 days after inoculation of hSC with the virus. Levels of viral RNA were lower than those in the highly permissive Huh7.5.1 cells, but corresponded to a viral load of 4.5 log and 3.9 log HCV copies intracellularly and in supernatants respectively (Figure 2A). The replication of HCV RNA could be inhibited by the addition of 10 μΜ HCV-796 (97.9% inhibition) or 1 μΜ VX-950 (74.2% inhibition) (Figure 4). Viral R A replication occurs on modified intracellular membranes, forming a membranous web. Swollen endoplasmatic reticulum (ER), double-membrane and multi-membrane vesicles were observed by means of electron microscopy in HCV-infected hSC-progeny (Data not shown).
Newly produced HCV is packed in very low density (VLDL)- and low density (LDL)- lipoviroparticles, consisting mainly of apolipoprotein B-100 (ApoBlOO). Albumin, ApoBlOO and HCV core protein was secreted in the supematants of infected hSC-progeny at a relatively constant rate over time, indicating stable replication (Figure 2B).
Ultimate proof that the entire replication cycle and formation of infectious particles can take place in hSC-progeny cultures comes from the observation that supernatant from infected cultures is able to result in infection of Huh7.5.1 cells. Indeed, supematants obtained at different periods following the initial inoculation contained infectious HCV virions capable of re-infecting Huh7.5.1 cells (Data not shown). Carry-over from the inoculum was excluded. In conclusion, we demonstrate here that the receptors for HCV entry are expressed on hSC hepatic progeny and that following inoculation of HCV, hepatic progeny secreted albumin, ApoBlOO and HCV virions, and that culture supematants support secondary infection of Huh7.5.1 cells. Based on our results, we estimate that about 5% of the hSC-progeny is susceptible to HCV. Interestingly, also in livers of patients (with a viral load > 5 log HCV) only a small fraction (7-20%) of hepatocytes is infected with HCV. Moreover, we show here and have shown before (Roelandt, P., et al. 2010) that a small fraction (1-5%) of hSC progeny express features of mature hepatocytes (ALB +/ AFP" cells). It is possible that cells other than hepatocytes are also infected. As has been shown for Huh7.5.1 cells and primary hepatocytes, infected cells in clusters were observed (Data not shown), suggestive for cell-to- cell HCV spread. The majority of the experiments was performed on hESC-H9 (n=6), but ApoBlOO and HCV core protein could also be detected in the supematants of hESC-Hl (n=l) and hiPSC-BJl (n=l), indicating that these findings are not restricted to the hESC-H9 cell line.
This is to the best of our knowledge the first report demonstrating productive infection with HCV of hepatocytes derived from human pluripotent stem cells. These findings form the basis for the development of a novel cell culture model for HCV replication that is physiological more relevant for the clinical situation than the use of hepatoma cell lines and may be complementary to and more useful than existing systems. Such cultures are possibly also infectable with clinical isolates/different genotypes. We believe that this model will be of great importance for further research on hepatitis virus, and more particularly HCV, as it is complementary to and more useful than existing systems. Furthermore, our findings will allow the study of HCV replication in a patient-specific cellular background by employing iPSC. This novel cell culture system will allow for the first time to study HCV in different subpopulations e.g. non-responders, relapsers, or IL28B-polymorphism.
EXAMPLE 2: Hepatitis A Virus infection
1. Cells and viruses
FPvhK-4 cells (ATCC CRL-1688) and BS-C-1 cells (ATCC CCL-26) were grown in Dulbecco's modified Eagle's medium (DMEM; Gibco, Gent, Belgium) supplemented with 10% fetal bovine serum (FBS; Integra, Leuvenheim, the Netherlands) in a humified 5% C02 incubator at 37°C. Hepatitis A virus (HAV) strain HM 175/18f (ATCC VR-1402, Lemon et al, 1991) was grown in BS-C-1 cells in DMEM with 2% FBS at 35°C. Virus was harvested by 3 freeze-thaw cycles followed by centrifugation at lOOOg for 10 minutes at 4°C.
Interferon Interferon-alpha 2b (IFNa, Intron-A®) was purchased from Schering Plough (Kenilworth, NJ), diluted to 3.105 international units (IU)/mL in phosphate-buffered saline (PBS; Lonza, Venders, Belgium) supplemented with 10% glycerol and 0.1 %> bovine serum albumin, stored at -80°C and kept at 4°C after thawing.
2. Virus titration
HM175/18f virus samples were titrated by end-point dilution. To this end, FRhK-4 cells were seeded in 96-well plates (BD Falcon; Franklin Lakes, NJ) at 2.104 cells per well in ΙΟΟμί of DMEM supplemented with 2% FBS and incubated at 37°C. After 24h, cells were confluent, medium was removed and ΙΟΟμί of a 1 : 10 virus dilution series in medium was added to each well. Each dilution was analyzed in 2-fold. Plates were incubated at 35°C for 7 days and subsequently scored by microscopy for cytopathic effect (CPE). The tissue culture infectious dose 50 (TCID50) corresponds to a viral dose sufficient to induce CPE in half of the cells in a tissue culture and was calculated by the method of Reed and Muench (Reed et al, 1938). 3. Infection of stem cell-derived hepatocytes
The differentiation protocol was performed for a 12-well plate as described. At 18 days post start of differentiation (d.p.s.d.), cells were transferred to 35°C. Pretreatment with IFNa at 300 IU/mL was started for 3 wells at 21 d.p.s.d.. The following day, medium was removed and to 3 non-treated and 3 IFNa-treated wells, ImL of a HM175/18f dilution in liver differentiation medium (LDM) supplemented with hepatocyte growth factor (HGF) was added, corresponding to 1.39xl05 TCID50 per well. Additionally, IFNa was added to the pretreated wells to a final concentration of 300 IU/mL. To 2 wells serving as cell control (CC), ImL of plain LDM with HGF was added. The cultures were incubated at 35°C for 4h. Afterwards, the inoculum was removed and cell layers were washed 5 times with plain DMEM. One mL of LDM with HGF was added to each well, IFN was added to the 3 pretreated wells and to 1 CC well. After lh, a 120μί sample was taken of each well for assessment of virus input and the cultures were further incubated at 35°C.
Every other day until 31 days post infection (d.p.i.), 750μί was removed from each well and replaced with ImL of fresh LDM with HGF, with or without IFNa. The removed medium was stored at -80°C for later analysis through reverse transcription quantitative PCR (RT- qPCR). Virus samples were titrated on FRhK-4 every week. At 31 d.p.i., medium was removed from all wells and cell layers were washed 3 times with PBS. Cells were lysed and RNA was extracted with the Qiagen RNeasy kit (Venlo, the Netherlands) according to the manufacturer's instructions.
Viral RNA was extracted from culture medium with the NucleoSpin RNA virus kit (Macherey-Nagel, Diiren, Germany) according to the manufacturer's instructions. Primers and probes for TaqMan-based quantification of HAV RNA were based on published sequences (Silberstein et al, 2003). As a forward primer 5'- GGCATTTAGGTTTTTCCTCATTCTTA-3 ' was used and the reverse primer was 5'- AATGTCTGCCAAAGACAGGATGT-3 ' . The TaqMan probe was labeled with 6- carboxyfluorescein (FAM) at the 5 ' end and with a minor groove binder (MGB) at the 3 ' end (5 ' -6F AM-C AAGGTATTTTCC AG ACTGTTGGGAGTGGTCT-MGBNFQ-3 '). Reactions were performed with One-Step qRT-PCR mix (Eurogentec, Seraing, Belgium) in a final volume of 25 containing 3μΜ of each primer, 67nM of probe and 5μΙ^ of RNA sample. PCR was performed using the ABI 7500 Fast Real-Time PCR System (Applied Biosystems; Foster City, CA) under following conditions: 30 min at 48°C and 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. Data were analyzed with ABI PRISM 7500 SDS software (version 1.3.1, Applied Biosystems). For absolute quantification, standard curves were generated using 10-fold dilutions of template preparations of known concentrations.
Cultures of stem cell-derived hepatocyte cultures were infectable with HAV (strain HM175/18f) (see Figure 5A). In the first 6 d.p.i. a rapid increase in viral titer was observed (4 logio increase) with a small decline of 1 logio afterwards. Up until 31 d.p.i., viral titers remained at a similar level, despite the partial medium changes every other day. When cultures were treated with IFNa continuously, viral replication was markedly suppressed (see Figure 5B). Infectivity of the viral progeny was confirmed by titration on FRhK-4 cells and microscopic evaluation of the produced CPE (data not shown).
EXAMPLE 3: Flavivirus infection
I. Methodology 1. Cells and viruses
DENV (Dengue Fever Virus) serotype 2 New Guinea C [DENV-2 NGC] were cultured on C6/36 mosquito cells (from Aedes albopictus; American Type Culture Collection at 28°C.
Yellow fever virus (YFV) 17D vaccine strain (Stamaril®) [Aventis Pasteur (MSD, Belgium)] was passaged once in Vero-B cells to prepare a working virus stock and stored at -80°C until further use. 2. Monitoring YFV and DENV replication.
The differentiation protocol was performed for a 12-well plate as described. At x days post start of differentiation (d.p.s.d) medium was removed and ImL of a diluted virus stock [either DENV-2 (NGC) or YFV-17D] in liver differentiation medium (LDM) supplemented with hepatocyte growth factor (HGF) was added. To 2 wells serving as cell control (CC), ImL of plain LDM with HGF was added. The cultures were incubated for 4h after which the inoculum was removed and cell layers were washed 5 times with plain DMEM to remove non-adsorbed virus and cultures were further incubated. One mL of LDM with HGF was added to each well. After lh, a sample was taken of each well for assessment of virus input and the cultures were further incubated. At x days post infection (d.p.i.), 750μί culture supernatant was removed from each well and replaced with lmL of fresh LDM with HGF, with or without IFNa. The removed medium was stored at -80°C for later analysis through reverse transcription quantitative PCR (RT-qPCR). Viral RNA load in supernatant was determined by quantitative reverse transcriptase-PCR (qRT-PCR). RNA was isolated from 150 μΐ supernatant with the NucleoSpin RNA virus kit (Macherey-Nagel, Germany) as described by the manufacturer. Primers and probe sequences are described in Kaptein et al, 2010. The TaqMan probe was fluorescently labeled with 6-carboxyfluorescein (FAM) at the 5' end as the reporter dye and with minor groove binder (MGB) at the 3' end as the quencher. One-step, quantitative RT-PCR was performed in a total volume of 25 μΐ, containing 13.9375 μΐ H20, 6.25 μΐ master mix (Eurogentec, Belgium), 0.375 μΐ forward primer, 0.375 μΐ reverse primer, 1 μΐ probe, 0.0625 μΐ reverse transcriptase (Eurogentec) and 3 μΐ sample. RT- PCR was performed using the ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Branchburg, NJ) using the following conditions: 30 min at 48°C and 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. The data was analyzed using the ABI PRISM 7500 SDS software (version 1.3.1; Applied Biosystems). For absolute quantification, standard curves were generated using 10-fold dilutions of template preparations of known concentrations. Viral antigen expression in infected cultures was monitored at x days post infection. Cells were stained with the anti-dengue E protein antibody (Ab) clone 3H5 (Millipore, Billerica, MA) or the anti-YFV NS1 Ab 1A5, and the secondary Ab Alexa Fluor 488 (Millipore). Following DAPI staining, the cultures were visualized using a confocal laser scanning microscope (LCSM, Leica Microsystems, Germany).
At day 4 after infection, a clear cytopathogenic effect is present in the Dengue Fever Virus infected cells (data not shown). These results clearly show that the stem cell derived hepatocytes as prepared by the method according to this invention, are also infectable with flaviviruses. EXAMPLE 4: Plasmodium infection
I. Background to the experiment
The malaria parasite exhibits a complex life cycle involving an insect vector and a vertebrate host. Four Plasmodium species infect humans: P. falciparum, P. vivax, P. ovale and P. malariae. All four species exhibit a similar life cycle with only minor variations. A proportion of the liver-stage parasites from P. vivax and P. ovale go through a dormant period instead of immediately undergoing asexual replication. These hypnozoites will reactivate several weeks to months (or years) after the primary infection and are responsible for relapses. The sporozoites are arrested by the circum sporozoite protein (CSP) on the sporozoit that interacts with thrombospondin-related anonymous protein; on hepatocytes, stellate cells and kupfer cells. These large proteoglycans protrude through fenestrations in LSECs into the liver sinusoids. TRAPs are very highly sulphated and therefore different from other tissues. In particular sulphation of the glycosaminoglycan chains at both the N- and O-positions is required for sporozoite adhesion to cells. It is believed that invasion in the liver occurs via kupfer cells: sporozoites invade kupfer cells and these infected kupfer cells pass through the endothelial barrier. Once in space of Disse, sporozoites invade sequentially multiple hepatocytes before invading and infecting the final hepatocyte. This requires specific lipases and pore forming proteins: sporozoite microneme protein essential for cell traversal (SPECT)l, SPECT2, cell traversal protein for ookinete and sporozoite (CelTOS) and the phospho lipase PbPL. The host ell wounding caused by the passage through different hepatocytes results in host cell wounding, which induces factors that increase the susceptibility of hepatocytes to infection, such as hepatocyte growth factor (HGF). As for hepatitis viruses, receptors on hepatocytes to which the sporozoites bind include CD81 and SRB1.
The intracellular parasite undergoes an asexual replication known as exoerythrocytic schizogony within the hepatocyte. Exoerythrocytic schizogony culminates in the production of merozoites. To multiply in the hepatocyte, a number of Plasmodium specific molecules are needed, termed upregulated in infective sporozoites (UIS)3 and 4, and Pb36p, and hepatocyte-specific APOA1 is also required. The release of Plasmodium merozoites from hepatocytes is usually referred to as occurring after hepatocyte rupture, even if this has not been seen, and it is unknown what mechanism underlies this. First the merozoites leave the parasitophorous vacuole, intermix with the cell cytoplasm and then are released as merozomes. The membrane is host cell derived and hence not seen by the immune system, allowing the merozoites to be transported to RBCs where they can infect. Infection of the liver causes no symptoms but is required for subsequent infection of RBS. Hence, mechanisms to prevent infection/replication of merozoites in the liver could stop infection and symptoms from blood cell death
Merozoites invade reticulocytes where they initially become larger. At that stage the detectable as 'ring form' because of its morphology. The trophozoite ingests host cytoplasm and causes degradation of hemoglobin into amino acids as food for the trophozoit. Subsequently the trophozoit undergoes several rounds of nuclear division without cytokinesis, resulting in the creation of the schizont. Merozoites the bud from the mature schizont, and the merozoites are released following rupture of the infected erythrocyte. Subsequently the merozoits can reinvade erythrocytes with a new blood-stage replicative cycle. The typical episodic fever is the result of synchronous lysis of infected erythrocytes. P. malariae exhibits a 72 hour periodicity, whereas the other three species exhibit 48 hour cycles. P. falciparum may also cause continuous fever and is responsible for more morbidity and mortality than the other species.
105 day 20 pluripotent stem cell derived hepatocyte progeny is seeded into collagen-coated wells of 24-well plates in hepatic differentiation medium. At 12-24 hours after seeding, P. Vivax or P. Falciparum sporozoites are isolated from the salivary glands of infected Anopheles mosquitoes and added to the Hepatocyte culture. After an incubation period of 2 hours the sporozoite-containing medium is removed and fresh medium is added. Production of merozoites in the hepatocytes is followed. After 3-7 days cells are analyzed to detect presence of parasitophorous vacuole, and merozomes containing merozoites. Cells are assessed for cell death. While the invention has been particularly shown and described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various alterations in form and detail, including the optimization to increase the efficiency of our system, may be made therein without departing from the spirit and scope of the invention, as defined by the appended claims.
All references cited herein are incorporated by reference for the teachings referred to in citing these references.
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