Maturation of hepatocyte-like cells derived from human pluripotent stem cells
10294457 · 2019-05-21
Assignee
Inventors
Cpc classification
C12N2501/999
CHEMISTRY; METALLURGY
C12N2501/385
CHEMISTRY; METALLURGY
C12N2506/45
CHEMISTRY; METALLURGY
C12N2501/06
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention relates to directed differentiation and maturation of hepatocyte-like cells. In particular, the present invention relates to exposure of hepatocyte-like cells to an activator of a retinoic acid responsive receptor, such as retinoic acid (RA), optionally in combination with an inhibitor of GSK-3 (Glycogen synthase kinase 3) or activator of Wnt signalling and/or with the overlay of the cells with one or more components characteristic of the mammalian extracellular matrix (matrix overlay). The present invention also relates to exposure of hepatocyte-like cells to an activator of a retinoic acid responsive receptor, such as retinoic acid (RA), optionally in combination with an inhibitor of a cycline dependent kinase (CDK) and/or with the overlay of the cells with one or more components characteristic of the mammalian extracellular matrix (matrix overlay). The hepatocyte-like cells obtained in accordance with the present invention show a phenotype which is more similar to that of primary hepatocytes than previously shown.
Claims
1. A method for promoting the maturation of in vitro derived human hepatocyte-like cells, the method comprising: exposing said human hepatocyte-like cells to an activator of a retinoic acid responsive receptor selected from the group consisting of 9-cis-retinoic acid, 13-cis-retinoic acid, SR11237, and combinations thereof, thereby promoting the maturation of said human hepatocyte-like cells by increasing the gene expression of one or more markers for mature hepatocytes selected from the group consisting of adult isoforms of HNF4, CYP1A2, CYP2B6, CYP2C9, CYP3A4, CYP3A5, CAR, GSTA1-1, NTCP and PXR; wherein the human hepatocyte-like cells exposed to said activator of a retinoic acid responsive receptor do not exhibit gene and protein expression of Oct4.
2. The method according to claim 1, further comprising culturing human hepatic progenitor cells, which do not exhibit gene and protein expression of Oct4, under differentiation conditions to obtain said hepatocyte-like cells.
3. The method according to claim 2, further comprising initially culturing human pluripotent stem (hPS) cells under differentiation conditions to obtain said hepatic progenitor cells.
4. The method according to claim 3, wherein the initial culturing of hPS cells includes culturing the hPS cells under differentiation conditions to obtain cells of the definitive endoderm (DE cells) and further culturing the obtained cells under differentiation conditions to obtain said hepatic progenitor cells.
5. The method according to claim 4, wherein the differentiating hPS cells are exposed to a DNA demethylating agent, and wherein the exposure to said DNA demethylating agent takes place during the differentiation of the hPS cells into DE cells.
6. The method according to claim 3, wherein the differentiating hPS cells are exposed to a DNA demethylating agent.
7. The method according to claim 2, wherein the hepatic progenitor cells are derived from human pluripotent (hPS) stem cells.
8. The method according to claim 2, wherein said differentiation conditions for obtaining hepatocyte-like cells are characterized by culturing said human hepatic progenitor cells in a differentiation medium comprising one or more growth factors and/or one or more differentiation inducers.
9. The method according to claim 1, further comprising exposing said hepatocyte-like cells to a GSK-3 inhibitor.
10. The method according to claim 9, further comprising exposing said hepatocyte-like cells to a CDK inhibitor.
11. The method according to claim 9, wherein the GSK-3 inhibitor is selected from the group consisting of: 9-Bromo-7, 12-dihydro-indolo [3,2-d][1]benzazepin-6(5H)-one, also known as Kenpaullone or NSC 664704; 1-Aza-Ken-paullone (9-Bromo-7,12-dihydro-pyrido[3,2:2,3]azepino[4,5-b]indol-6(5H)-one); Alsterpaullone (9-Nitro-7,12-dihydroindolo-[3,2-d][1]benzazepin-6(5)-one); BIO (2Z,3E)-6-Bromoindirubin-3-oxime (GSK-3 Inhibitor IX); BIO-Acetoxime (2Z,3E)-6-Bromoindirubin-3-acetoxime (GSK-3 Inhibitor X); (5-Methyl-IH-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine (GSK-3 Inhibitor XIII); Pyridocarbazole-cyclopenadienylruthenium complex (GSK-3 Inhibitor XV); TDZD-8 4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (GSK-3beta Inhibitor I); 2-Thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole (GSK-3beta Inhibitor II); OTDZT 2,4-Dibenzyl-5-oxothiadiazolidine-3-thione (GSK-3beta Inhibitor III); alpha-4-Dibromoacetophenone (GSK-3beta Inhibitor VII); AR-AO 14418 N-(4-Methoxybenzyl)-N-(5-nitro-1,3-thiazol-2-yl)urea (GSK-3beta Inhibitor VIII); 3-(1-(3-Hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrrole-2,5-dione (GSK-3beta Inhibitor XI); TWS119 pyrrolopyrimidine compound (GSK-3beta Inhibitor XII); L803 H-KEAPPAPPQSpP-NH.sub.2 or its Myristoylated form (GSK-3beta Inhibitor XIII); 2-Chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone (GSK-3beta Inhibitor VI); Aminopyrimidine CHIR99021; 3-(2,4-Dichlorophenyl)-4-(1-methyl-1 H-indol-3-yl)-1 H-pyrrole-2,5-dione (SB216763); and Indirubin-3-monoxime.
12. The method according to claim 9, wherein the GSK-3 inhibitor further exhibits inhibitory activity towards a cyclin dependent kinase (CDK).
13. The method according to claim 9, wherein the GSK-3 inhibitor further exhibits inhibitory activity towards cyclin dependent kinase 2 (CDK2).
14. The method according to claim 1, further comprising exposing said hepatocyte-like cells to an activator of Wnt signaling.
15. The method according to claim 14, wherein the activator of Wnt is a Wnt protein.
16. The method according to claim 1, further comprising exposing said hepatocyte-like cells to a CDK inhibitor.
17. The method according to claim 1, further comprising exposing said hepatocyte-like cells to a matrix overlay simultaneous to exposure to the activator of a retinoic acid responsive receptor.
18. The method according to claim 1, further comprising exposing said hepatocyte-like cells to a GSK-3 inhibitor and a matrix overlay simultaneous to exposure to the activator of a retinoic acid responsive receptor.
19. The method according to claim 1, further comprising exposing said hepatocyte-like cells to an activator of Wnt signaling and a matrix overlay simultaneous to exposure to the activator of a retinoic acid responsive receptor.
20. The method according to claim 1, further comprising exposing said hepatocyte-like cells to a CDK inhibitor and a matrix overlay simultaneous to exposure to the activator of a retinoic acid responsive receptor.
21. The method according to claim 1, wherein the activator of a retinoic acid responsive receptor is 9-cis-retinoic acid, 13-cis-retinoic acid, or a combination of 9-cis-retinoic acid and 13-cis-retinoic acid.
22. The method according to claim 1, wherein the activator of a retinoic acid responsive receptor is 9-cis-retinoic acid.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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(53) FIG. 13A1. Morphology of hESC-derived definitive endodermal cells (derived with basic protocol C) without a 5-aza-deoxycytidine treatment during the pre-endodermal phase (day 0-7 of the protocol).
(54) FIG. 13A2. Morphology of hESC-derived definitive endodermal cells (derived with basic protocol C) with a 5-aza-deoxycytidine treatment during the pre-endodermal phase (day 0-7 of the protocol).
(55) FIG. 13B1. Morphology of hiPSC-derived definitive endodermal cells (derived with basic protocol D) without a 5-aza-deoxycytidine treatment during the pre-endodermal phase (day 0-7 of the protocol).
(56) FIG. 13B2. Morphology of hiPSC-derived definitive endodermal cells (derived with basic protocol D) with a 5-aza-deoxycytidine treatment during the pre-endodermal phase (day 0-7 of the protocol).
(57) FIG. 13C1. Oct4-immunstaining and DAPI nuclear staining of hiPSC-derived definitive endodermal cells (derived with basic protocol D) without a 5-aza-deoxycytidine treatment during the pre-endodermal phase (day 0-7 of the protocol).
(58) FIG. 13C2. Oct4-immunstaining and DAPI nuclear staining of hiPSC-derived definitive endodermal cells (derived with basic protocol D) with a 5-aza-deoxycytidine treatment during the pre-endodermal phase (day 0-7 of the protocol).
(59) FIG. 13D1. mRNA expression of stem cell marker Oct4 in hESC- and hiPSC-derived definitive endodermal cells (derived with basic protocols C and D, respectively) with and without a 5-aza-deoxycytidine treatment during the pre-endodermal phase (day 0-7 of the protocol).
(60) FIG. 13D2. mRNA expression of stem cell marker Nanog in hESC- and hiPSC-derived definitive endodermal cells (derived with basic protocols C and D, respectively) with and without a 5-deoxycytidine treatment during the pre-endodermal phase (day 0-7 of the protocol).
(61) FIG. 13D3. mRNA expression of DE marker Sox17 in hESC- and hiPSC-derived definitive endodermal cells (derived with basic protocols C and D, respectively) with and without a 5-deoxycytidine treatment during the pre-endodermal phase (day 0-7 of the protocol).
(62) FIG. 13D4. mRNA expression of DE marker Cxcr4 in hESC- and hiPSC-derived definitive endodermal cells (derived with basic protocols C and D, respectively) with and without a 5-deoxycytidine treatment during the pre-endodermal phase (day 0-7 of the protocol).
(63) FIG. 13D5. mRNA expression of DE marker FoxA2 in hESC- and hiPSC-derived definitive endodermal cells (derived with basic protocols C and D, respectively) with and without a 5-deoxycytidine treatment during the pre-endodermal phase (day 0-7 of the protocol).
(64) FIG. 13D6. mRNA expression of DE marker hHEX in hESC- and hiPSC-derived definitive endodermal cells (derived with basic protocols C and D, respectively) with and without a 5-deoxycytidine treatment during the pre-endodermal phase (day 0-7 of the protocol).
(65) FIG. 13D7. mRNA expression of extraembryonic marker Sox7 in hESC- and hiPSC-derived definitive endodermal cells (derived with basic protocols C and D, respectively) with and without a 5-deoxycytidine treatment during the pre-endodermal phase (day 0-7 of the protocol).
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(98) FIG. 21A1. Functional expression of CYP2C9 enzyme in hiPSC-derived hepatocyte-like cells (derived with basic protocol D) treated early with 5-deoxycytidine and exposed late to 9cis retinoic acid, Kenpaullone or an analogue to Kenpaullone.
(99) FIG. 21A2. Functional expression of CYP 3A enzyme in hiPSC-derived hepatocyte-like cells (derived with basic protocol D) treated early with 5-deoxycytidine and exposed late to 9cis retinoic acid, Kenpaullone or an analogue to Kenpaullone.
(100) FIG. 21B1. Functional expression of CYP2C9 enzyme in hESC-derived hepatocyte-like cells (derived with basic protocol C) treated early with 5-deoxycytidine and exposed late to 9cis retinoic acid, Kenpaullone or an analogue to Kenpaullone.
(101) FIG. 21B2. Functional expression of CYP3A enzyme in hESC-derived hepatocyte-like cells (derived with basic protocol C) treated early with 5-deoxycytidine and exposed late to 9cis retinoic acid, Kenpaullone or an analogue to Kenpaullone.
EXAMPLES
(102) Examples of general culturing and passaging techniques are disclosed in applications WO2004/099394, WO2003/055992, WO/2007/042225, WO2007/140968 and WO2011116930.
(103) As laid out in the following examples, the starting material may comprise any hepatic progenitor cell type, particularly one derived through an initial differentiation towards a definitive or extraembryonic lineage from a human pluripotent stem cell. The starting material may also be any cell of hepatic progenitor lineage.
Example 1
Maintenance of hPS Cell Types
(104) All hPS cells (as defined above) can be used as staring material for this invention. For the examples below in particular hepatocyte-like cells were derived in vitro from undifferentiated human embryonic stem cells (hESC) established on mEF feeder cells (Heins et al 2004) and maintained under feeder-free conditions. The cell lines used for this experiment could be, but are not limited to the hES cell lines SA167, SA181, SA461 (Cellartis AB, Goteborg, Sweden) and they can be propagated as described by Heins et al. 2004. These cell lines are listed in the NIH stem cell registry, the UK Stem Cell bank and the European hESC registry and are available on request.
(105) Along with hPS obtained from hESC, hiPS (human induced pluripotent stem) cells have also been used for the derivation of hepatocytes for the examples of this invention.
(106) The hiPSC line used in this invention was derived as followed: Human dermal fibroblasts (CRL2429, ATCC) were maintained in DMEM supplemented with 10% fetal bovine serum, 1 glutamax, 5 U/ml penicillin and 5 g/ml streptomycin at 37 C. in a humidified atmosphere of 5% CO.sub.2 in air. Fibroblasts were tranduced with recombinant lentiviruses encoding mouse Oct4, Sox2, Klf4 and c-myc and cultured for 5 days. The transduced cells were then dispersed with trypsin and seeded onto mitomycin C treated human dermal fibroblast feeder cells at a density of 510.sup.3 cells/cm.sup.2 in their normal growth medium. After 24 hours the medium was replaced with knockout DMEM supplemented with 20% knockout serum replacement, 1 non-essential amino acids, 1 glutamax, 5 U/ml penicillin, 5 g/ml streptomycin, 100 M 2-mercaptoethanol and 30 ng/ml bFGF at 37 C. in a humidified atmosphere of 5% CO.sub.2 in air. Half of the volume of medium was replaced every day and colonies of iPS cells emerged after approximately 30 days. iPS colonies were picked, expanded in DEF-CS, and cell banks prepared. The banked cells were then characterised to check for the expression of endogenous Oct4, Sox2, Klf4 and c-Myc, silencing of transgenes, potential to differentiate into cell types representative of all three germ layers in vitro, and to confirm their authenticity by STR profiling and comparison with the parental fibroblast cell line (ATCC). Alternatively to reprogramming using lentivirus, hiPSC lines can also be reprogrammed using retrovirus, Sendai virus, adenovirus, episomal plasmid vectors, proteins and mRNAs or other techniques. Other suitable cell lines for use are those established by Chung et al. (2008), such as cell lines MA126, MA127, MA128 and MA129 (Advanced Cell Technology, Inc. Worcester, Mass., USA), which all are listed with the International stem cell registry. These cell lines have been derived (or obtained) without destruction of the human embryo by employing a single blastomere removal technique.
Example 2
Differentiation of hPS Cell Types to Produce Hepatocyte-Like
(107) Hepatocyte-like cells may be derived from hPS cells by employing the following exemplary basic protocols A, B, C, and D:
(108) Protocol A:
(109) Undifferentiated hPS cells are dissociated and seeded directly in freshly prepared day 0medium. The different mediums were prepared freshly and added day 0, 1, 2, 3, 4, 5, 7 and then every second or third day during the pre-hepatic phase, and differentiation and maturation phase.
(110) Day 0
(111) RPMI 1640 (+0.1% PEST, +1% Glutamax)
(112) 1B27
(113) 100 ng/ml Activin A
(114) 1 mM NaB
(115) 5 M ROCK inhibitor
(116) Day 1
(117) RPMI 1640 (+0.1% PEST, +1% Glutamax)
(118) 1B27
(119) 100 ng/ml Activin A
(120) 1 mM NaB
(121) Day 2-7
(122) RPMI 1640 (+0.1% PEST+1% Glutamax)
(123) 1B27
(124) 100 ng/ml Activin A
(125) 0.5 mM NaB
(126) On day 7 the cells are passaged. The cells are incubated for 3-7 minutes with TrypLE Select at 37 C., the same volume of VitroHES is added and the cell suspension is centrifuged at 200-300 g, 5-6 min. Thereafter, the cells are replated onto a Gelatine-based coating at a cell density of 50 000-350 000 cells/cm.sup.2 such as e.g. 100 000-300 000 cells/cm.sup.2, preferably 150 000 cells/cm.sup.2.
(127) Day7-14 (pre-hepatic)
(128) VitroHES
(129) 1% DMSO
(130) Day 14-45 (differentiation and maturation)
(131) WME+SQ (-GA1000)+1% Glutamax+0.1% PEST
(132) 0.1 M DexM
(133) 10 ng/ml OsM
(134) 20 ng/ml HGF
(135) 0.5% DMSO
(136) 1.4 M (2Z,3)-6-Bromoindirubin-3-oxime (BIO)
(137) Protocol B:
(138) Undifferentiated hPS cells are dissociated and seeded directly in freshly prepared day 0medium. The different mediums were prepared freshly and added day 0, 1, 2, 3, 4, 5, 7 and then every second or third day during the pre-hepatic phase, and differentiation and maturation phase.
(139) Day 0
(140) RPMI 1640 (+0.1% PEST, +1% Glutamax)
(141) 1B27
(142) 100 ng/ml Activin A
(143) 1 mM NaB
(144) 5 M ROCK inhibitor
(145) Day 1
(146) RPMI 1640 (+0.1% PEST, +1% Glutamax)
(147) 1B27
(148) 100 ng/ml Activin A
(149) 1 mM NaB
(150) 3 M CHIR99021
(151) Day 2-7
(152) RPMI 1640 (+0.1% PEST+1% Glutamax)
(153) 1B27
(154) 100 ng/ml Activin A
(155) 0.5 mM NaB
(156) On day 7 the cells are passaged. The cells are incubated for 3-7 minutes with TrypLE Select at 37 C., the same volume of VitroHES is added and the cell suspension is centrifuged at 200-300 g, 5-6 min. Thereafter, the cells are replated onto a Fibronectin-based coating at a cell density of 50 000-350 000 cells/cm.sup.2 such as e.g. 100 000-300 000 cells/cm.sup.2, preferably 150 000 cells/cm.sup.2. For media d7-14 (pre-hepatic) and 14-45 (differentiation and maturation) see Protocol A.
(157) Protocol C:
(158) Undifferentiated hPS cells are dissociated and seeded directly in freshly prepared day 0medium. The different mediums were prepared freshly and added day 0, 1, 2, 3, 4, 5, 7 and then every second or third day during the pre-hepatic phase, and differentiation and maturation phase. The pre-treatment medium is available from Cellectis AB (Arvid Wallgrens Backe 20, 41346 Gothenburg, Sweden).
(159) Day 0
(160) Pre-treatment medium
(161) 3 M CHIR99021
(162) 5 M ROCK inhibitor
(163) Day 1
(164) Pre-treatment medium
(165) 3 M CHIR99021
(166) Day 2
(167) RPMI 1640 (+0.1% PEST+1% Glutamax)
(168) 1B27
(169) 50 ng/ml Activin A
(170) 3 M CHIR99021
(171) 5 M LY294002
(172) Day 3
(173) RPMI 1640 (+0.1% PEST+1% Glutamax)
(174) 1B27
(175) 50 ng/ml Activin A
(176) 5 M LY294002
(177) Day 4-7
(178) RPMI 1640 (+0.1% PEST+1% Glutamax)
(179) 1B27
(180) 50 ng/ml Activin A
(181) For passage d7, media d7-14 (pre-hepatic) and 14-45 (differentiation and maturation) see Protocol B.
(182) Protocol D:
(183) Undifferentiated hPS cells are dissociated and seeded directly in freshly prepared day 0medium. The different mediums were prepared freshly and added day 0, 1, 2, 3, 4, 5, 7 and then every second or third day during the pre-hepatic phase, and differentiation and maturation phase. The pre-treatment medium is available from Cellectis AB (Arvid Wallgrens Backe 20, 41346 Gothenburg, Sweden).
(184) Day 0
(185) Pre-treatment medium
(186) 3 M CHIR99021
(187) 5 M ROCK inhibitor
(188) Day 1
(189) RPMI 1640 (+0.1% PEST+1% Glutamax)
(190) 1B27
(191) 50 ng/ml Activin A
(192) 3 M CHIR99021
(193) 5 M LY294002
(194) Day 2
(195) RPMI 1640 (+0.1% PEST+1% Glutamax)
(196) 1B27
(197) 50 ng/ml Activin A
(198) 5 M LY294002
(199) Day 3-7
(200) RPMI 1640 (+0.1% PEST+1% Glutamax)
(201) 1B27
(202) 50 ng/ml Activin A
(203) For passage d7, media d7-14 (pre-hepatic) and 14-45 (differentiation and maturation) see Protocol B.
Example 3
Effect of Treatment of hESC-Derived Hepatocyte-Like Cells with 9 Cis-Retinoic Acid on Expression of Different HNF4 Isoforms
(204) Procedure:
(205) Following the basic protocol A, hepatocyte-like cells derived from hES cells cultured on a Gelatin-based coating were treated with 1 or 2 M 9 cis-retinoic acid for 5 hr, 24 hr or 48 hr on day 23 of the protocol (i.e. on day 9 of the differentiation and maturation) or long term from day 14 (i.e. starting day 1 of the differentiation and maturation phase) and onwards and analysed immediately after the exposure (
(206) Results:
(207) A) 5 hr RA-exposure strongly increase expression of the adult HNF4a1-3 isoforms, but also increase the expression of the fetal HNF4a4-9 isoforms. 24-, 48 hr-exposure and continuous RA-treatment slightly increase the adult HNF4a1-3 isoforms and slightly decrease the fetal HNF4a4-9 isoforms making the ratio of 1-3 isoforms/4-9 isoforms more similar to hphep, see also
(208) B) Human primary hepatocytes (hp hep) have a high ratio of the adult HNF4a isoforms 1-3 to the fetal 4-9 isoforms, whereas HepG2 have a low ratio. 24 hr and 48 hr RA-exposure and long term/continuous treatment increase the 1-3/4-9 ratio of hESC-derived hepatocytes to levels similar as in hp hep. 5 hr exposures do not increase the 1-3/4-9 ratio since also expression of 4-9 isoforms increases (see
(209) C) A 5 hr exposure with 1 M RA increases the expression of HNF4 isoforms 1-3 immediately after the exposure (see also A), but 7 days later the expression of isoforms 1-3 is slightly lower in the RA-treated cells than in the untreated control cells. The expression of isoforms 4-9 is slightly lower in the RA-treated cells than in the control immediately after the exposure. 7 days later expression of fetal isoform is raised over control values.
(210) Therefore the optimal culture conditions for producing an increase in the adult isoforms of HNF4a with minimal increase or decrease in fetal isoforms on day 23 involve the continuous treatment or 24 hr or 48 hr exposures of 1 or 2 M RA on d23, corresponding to an expression profile closest to that of primary human hepatocytes (hp hep). The skilled person wishing to produce cells with an unchanged expression profile might instead select a 5 hr exposure to RA.
Example 4
Effect of Treatment of hESC-Derived Hepatic Progenitors and Hepatocyte-Like Cells with 9 Cis-Retinoic Acid (RA) on CYP Activity
(211) Procedure:
(212) Following the basic protocol A, differentiating hES cell derived hepatic progenitors and hepatocyte-like cells cultured on a Gelatin-based coating were treated with 1 M 9-cis retinoic acid for 5, 24 or 48 hr exposures on days 21, 22 or 23 of the protocol (i.e. on day 7, 8 or 9 of the differentiation and maturation phase;
(213) Immediately after end of the RA treatment, the cell cultures are subjected to a CYP activity assay according to the following protocol: Cells are washed twice with warm Williams medium E w/o phenol red (+0.1% PEST). Then CYP activity assay, consisting of warm Williams medium E w/o phenol red (+0.1% PEST), 2 mM L-Glutamine, 25 mM HEPES, 26 M Phenacetin (model substrate for CYP1A), 9 M Diclofenac (model substrate for CYP2C9) and 3 M Midazolam (model substrate for CYP3A), is added to the cells (e.g. 220 l/24 well) and incubated for 16 hr at 37 C. Then supernatant is collected and centrifuged for 20 min at 10.000 rcf at 4 C. Subsequently, 120 l of the supernatant is transferred into a 96 well plate which is sealed with a tight seal tape and stored at 20 or 80 C. until LC/MS-analysis of metabolite formation: Acetaminophen (Paracetamol) for CYP1A, OH-Diclofenac for CYP2C9 and OH-Midazolam for CYP3A.
(214) Results:
(215) A) After 5 and 24 hr RA-exposures on day 21 of the protocol (i.e. on day 7 of the differentiation and maturation phase), an immediate increase of CYP1A and 3A activity in hESC-derived hepatocytes can be observed: CYP1A activity is on same level as in primary hepatocytes cultured for 48 hr, whereas CYP3A activity is roughly 25% of primary hepatocytes cultured for 48 hr. HepG2 have much lower CYP1A and 3A activity than hESC-derived hepatocytes. On day 23 in the protocol no CYP2C9 activity could be detected in hESC-derived hepatocytes.
(216) B) 7 days after a single 5 hr RA-exposure on day 23 of the protocol (i.e. on day 9 of the differentiation and maturation phase) hESC-derived hepatocytes still have increased CYP1A, 2C9 and 3A activities compared with untreated cells. However the increase in CYP1A and 2C9 activity is higher after 5 repeated 5 hr exposures (see
(217) C) 5 repeated 5 hr RA pulses on days 11, 16, 23, 25 and 30 of the protocol (i.e. on day 4 of the hepatic progenitor step and days 2, 9, 11 and 16 of the maturation step) lead to a significant increase of CYP1A-, 2C9- and 3A-activity on day 31 of the protocol. However, on day 24 of the protocol an increase of CYP1A-, but not of CYP3A-activity could be observed (no CYP2C9-activity detectable on day 24 of the protocol). Thus, repeated 5 hr RA-exposures have a stronger increasing effect on CYP1A and 2C9 activity than one single 5 hr RA-exposure (comp. to
(218) D) A comparison of 5, 24, 48 hr exposures and continuous treatment shows that the strongest increase of CYP3A activity is obtained with continuous RA-treatment compared to 5, 24 and 48 hr RA-exposures whereas the strongest increase of CYP1A activity is observed after a 24 hr RA-exposure.
(219) The strongest increase in expression of CYP2C9 is observed with repeated 5 hr exposure commencing on d24 (i.e. day 12 of the differentiation and maturation phase). Therefore the skilled person wishing to effect an increase in a particular CYP gene may select from these pulse conditions according to their gene of interest.
Example 5
Treatment with 9 Cis-Retinoic Acid (RA) Induces a More Adult Phenotype in hESC-Derived Hepatocyte-Like Cells
(220) Procedure:
(221) Following the basic protocol A, hES cell derived hepatocyte-like cells cultured on a Gelatin-based coating were treated with 1 M 9-cis retinoic acid for 5 hr on day 21 of the protocol (i.e. on day 7 of the differentiation and maturation phase) or 24 hr on day 22-23 of the protocol (i.e. on day 8-9 of the differentiation and maturation phase).
(222) Cells were harvested on day 21 or 23 of the protocol (i.e. on day 7 or 9 of the differentiation and maturation phase) and gene expression was analysed using qRT-PCR, normalised to the house-keeping gene CREBBP and the results presented as relative quantification normalised to a calibrator (
(223) Results:
(224) A) An increase of mRNA expression of the adult hepatic gene CYP3A4 is observed immediately after a 5 RA-exposure on day 21 (i.e. on day 7 of the differentiation and maturation phase) as well as 2 and 7 days later (on day 23 and 30 of the protocol, respectively), Also a 24 hr RA-exposure on day 22-23 of the protocol (i.e. on day 8-9 of the differentiation and maturation phase) lead to an immediate up-regulation of adult CYP3A4 expression on day 23 (i.e. on day 9 of the differentiation and maturation phase).
(225) B) A 5 hr exposure on day 21 (i.e. on day 7 of the differentiation and maturation phase) decreases expression of the fetal hepatic gene CYP3A7 slightly immediately after the exposure on day 21 and strongly 2 days later on day 23 of the protocol (i.e. on day 9 of the differentiation and maturation phase). Similarly, a 24 hr RA-exposure on day 22-23 of the protocol (i.e. on day 8-9 of the differentiation and maturation phase) strongly decreases expression of the fetal hepatic gene CYP3A7 immediately after the exposure on day 23 of the protocol (i.e. on day 9 of the differentiation and maturation phase).
(226) C) A 5 hr exposure on day 21 (i.e. on day 7 of the differentiation and maturation phase) increases mRNA expression of the adult hepatic gene PXR on day 23 (i.e. on day 9 of the differentiation and maturation phase), but not immediately after the 5 hr exposure on day 21 (i.e. on day 7 of the differentiation and maturation phase).
(227) A 24 hr RA-exposure on day 22-23 of the protocol (i.e. on day 8-9 of the differentiation and maturation phase) also increases mRNA expression of the adult hepatic gene PXR expression.
(228) D,E) Continuous/long-term RA treatment leads to higher increase of mRNA expression of the adult hepatic gene CAR (D) and a stronger down-regulation of the fetal hepatic gene -Fetoprotein (AFP, E) than 5 and 24 hr RA exposures on d22 and day 21-22, respectively (day8 and day 7-8 of the differentiation and maturation phase, respectively).
(229) In this case it can be seen that exposure to RA at d21 (at the end of the hepatic progenitor phase) leads to an increase in the expression of adult genes CYP3A4, CAR and PXR and a decrease in fetal genes AFP and CYP3A7, thus showing that a more mature and adult phenotype is achieved. The skilled person can further refine this method by selecting 5 hr pulse, 24 hours pulse or continuous treatment if there is one specific gene or group of genes from within this set which they wish to up or down regulate.
Example 6
Effect of Treatment of hESC- and hiPSC-Derived Hepatocyte-Like Cells with 9 Cis-Retinoic Acid (RA), Kenpaullone (K) and a Thin Fibronectin-Collagen I-Overlay (thin FC-Overlay) on CYP Activity
(230) Procedure:
(231) Following the basic protocol B, differentiating hES cell derived hepatic progenitor cells and hepatocyte-like cells cultured on a Fibronectin-based coating were treated with continuous/long term treatment with 0.2 M 9cis-retinoic acid and 0.5 M Kenpaullone starting on day 14 of the protocol (i.e. day 1 of the differentiation and maturation phase) and received thin Fibronectin-Collagen I-overlays on day 14 and 16 of the protocol (i.e. day 1 and 3 of the differentiation and maturation phase) and is refreshed thereafter once a week on day 23, 30, 37 and so on (i.e. on day 9, 16, 23 and so on of the differentiation and maturation phase). This combination of thin Fibronectin-Collagen I-overlay, RA and Kenpaullone is called henceforth RA+matrix overlay+Kenpaullone.
(232) The thin Fibronectin-Collagen 1-overlay is applied as following: Prepare a 3 mg/ml rat tail Collagen I-solution by diluting the Collagen I stock with 0.02M acetic acid. Pre-warm the cell culture medium to room temperature and add 8 l of the 3 mg/ml Collagen I-solution per ml medium (=25 g Collagen I/ml) and 50 l of a 1 mg/ml Fibronectin solution per ml medium (=50 g Fibronectin/ml). Remove the old medium from the cultures and add 0.5 ml of the Collagen I and Fibronectin-containing medium per cm.sup.2 culture surface (=12.5 g Collagen I/cm.sup.2 and 25 g Fibronectin/cm.sup.2). For refreshing the overlay once a week, add 4 l of the 3 mg/ml Collagen I-solution per ml medium (=6.25 g/ml) and 10 l of a 1 mg/ml Fibronectin solution per ml medium (=5 g/ml).
(233) For analysing functional expression of CYP enzymes, the cell cultures are subjected to a CYP activity assay according to the following protocol: Cells are washed twice with warm Williams medium E w/o phenol red (+0.1% PEST). Then CYP activity assay, consisting of warm Williams medium E w/o phenol red (+0.1% PEST), 2 mM L-Glutamine, 25 mM HEPES, 26 M Phenacetin (model substrate for CYP1A), 9 M Diclofenac (model substrate for CYP2C9) and 3 M Midazolam (model substrate for CYP3A), is added to the cells (e.g. 220 l/24 well) and incubated for 16 hr at 37 C. Then supernatant is collected and centrifuged for 20 min at 10.000 rcf at 4 C. Subsequently, 120 l of the supernatant is transferred into a 96 well plate which is sealed with a tight seal tape and stored at 20 or 80 C. until LC/MS-analysis of metabolite formation: Acetaminophen (Paracetamol) for CYP1A, OH-Diclofenac for CYP2C9 and OH-Midazolam for CYP3A,
(234) Results:
(235) The inventors have found that, further to the use of RA alone, the combination of continuous/long term treatment with a thin Fibronectin-Collagen I-overlay, 0.5 M Kenpaullone, and 0.2 M RA [henceforth RA+matrix overlay+Kenpaullone] (starting on day 14 and onwards, i.e. starting on day 1 of the differentiation and maturation phase) reproducibly increases CYP activity in hESC-derived hepatocytes (
(236) A) The combination of continuous treatment of hESC-derived hepatocytes treated with 0.2 M RA and 0.5 M Kenpaullone (starting on day 14 and onwards) and the application of a thin Fibronectin-Collagen I-overlay is the only experimental group which has both increased CYP2C9- and 3A-activity on day 36 of the protocol (i.e. day 22 of the differentiation and maturation phase). However, some single or double treated groups also showed increase of CYP2C9- and 3A-activity, but none had both high CYP2C9- and 3A-activity. CYP1A activity is highest with overlay alone. HepG2 only show CYP1A activity and no 2C9 or 3A activity.
(237) B,C) The combination of continuous/long term treatment with a thin Fibronectin-Collagen I-overlay, 0.5 M Kenpaullone, and 0.2 M RA (starting on day 14 and onwards) reproducibly increases CYP1A, 2C9 and 3A activity in hESC-derived and hiPSC-derived hepatocytes.
(238) The expression levels of various CYP genes and other markers associated with a mature hepatic phenotype have been further examined in the following examples, which provide more detailed guidance for those wishing to improve mature hepatic phenotype using this triple combination.
Example 7
Increase in Expression of Hepatic Phase I and Phase II Enzymes, Drug Transporters and Nuclear Receptors in Hepatocyte-Like Cells
(239) Procedure:
(240) Following the basic protocols B (
(241) The thin Fibronectin-Collagen I-overlay is applied as following: Prepare a 3 mg/ml rat tail Collagen I-solution by diluting the Collagen I stock with 0.02M acetic acid. Pre-warm the cell culture medium to room temperature and add 8 l of the 3 mg/ml Collagen I-solution per ml medium (=25 g Collagen I/ml) and 50 l of a 1 mg/ml Fibronectin solution per ml medium (=50 g Fibronectin/ml). Remove the old medium from the cultures and add 0.5 ml of the Collagen I and Fibronectin-containing medium per cm.sup.2 culture surface (=12.5 g Collagen I/cm.sup.2 and 25 g Fibronectin/cm.sup.2). For refreshing the overlay once a week, add 4 l of the 3 mg/ml Collagen I-solution per ml medium (=6.25 g/ml) and 10 l of a 1 mg/ml Fibronectin solution per ml medium (=5 g/ml).
(242) Cells were harvested on day 23, 30 and 36/37 of the protocol (i.e. on day 9, 16 and 22/23 of the differentiation and maturation phase) and gene expression was analysed using qRT-PCR, normalised to the house-keeping gene CREBBP, and the results presented as relative quantification normalised to a calibrator (
(243) Results:
(244)
(245)
(246)
(247) The combination of Kenpaullone, matrix overlay and RA shows a synergistic effect over exposures with RA alone (see e.g. CYP3A4 mRNA expression in
(248) The skilled person may therefore select from a number of sources of pluripotent stem cell lines as starting material to implement the invention. The skilled person may also employ the results obtained in
Example 8
Increase in Expression of Hepatic Phase I and Phase II Enzymes, Drug Transporters and Nuclear Receptors in Hepatocyte-Like Cells: Pre-Exposure to DNA Demethylation Agent
(249) Procedure:
(250) Following the basic protocols C (hESC-derived hepatocytes) or D (hiPSC-derived hepatocytes), differentiating hES and hiPS cells in the pre-endodermal phase were treated with 10 nM of the DNA demethylating agent 5-aza-2-deoxycytidine for 24 hr on day 2 to 3 of the protocol.
(251) Later in the protocol, hPS cell derived hepatic progenitor cells and hepatocyte-like cells cultured on a Fibronectin-based coating were treated with continuous/long term treatment with 0.2 M 9cis-RA and 0.5 M Kenpaullone starting on day 14 of the protocol (i.e. on day 1 of the differentiation and maturation phase) and received thin Fibronectin-Collagen I-overlays on day 14 and 16 of the protocol (i.e. on day 1 and 3 of the differentiation and maturation phase) and is refreshed thereafter once a week on day 23, 30, 37 and so on (i.e. on day 9, 16, 23 and so on of the differentiation and maturation phase).
(252) The thin Fibronectin-Collagen I-overlay is applied as following: Prepare a 3 mg/ml rat tail Collagen I-solution by diluting the Collagen I stock with 0.02M acetic acid. Pre-warm the cell culture medium to room temperature and add 8 l of the 3 mg/ml Collagen I-solution per ml medium (=25 g Collagen I/ml) and 50 l of a 1 mg/ml Fibronectin solution per ml medium (=50 g Fibronectin/ml). Remove the old medium from the cultures and add 0.5 ml of the Collagen I and Fibronectin-containing medium per cm.sup.2 culture surface (=12.5 g Collagen I/cm.sup.2 and 25 g Fibronectin/cm.sup.2). For refreshing the overlay once a week, add 4 l of the 3 mg/ml Collagen I-solution per ml medium (=6.25 g/ml) and 10 l of a 1 mg/ml Fibronectin solution per ml medium (=5 g/ml).
(253) For analysis of mRNA expression (
(254) For analysing functional expression of CYP enzymes on day 36 (i.e. on day 22 of the maturation step;
(255) Results:
(256)
(257)
(258)
(259)
(260) The general trend seen here is that both hiPS and hESC-derived hepatocytes show increase in expression of mature markers; with treatment generating a small increase initially (d29) and a larger increase by d36. For example,
Example 9
Stable CYP Expression in Hepatocyte-Like Cells Derived from hESC and hiPSC Treated with a Demethylating Agent, Retinoic Acid, Kenpaullone and a Matrix Overlay
(261) Procedure:
(262) Following the basic protocols C (hESC-derived hepatocytes) or D (hiPSC-derived hepatocytes), cells in the DE-step were treated with 10 nM of the DNA demethylating agent 5-aza-2-deoxycytidine for 24 hr on day 2 to 3 of the protocol. Later in the protocol, hPS cell derived hepatic progenitor cells and hepatocyte-like cells cultured on a Fibronectin-based coating were treated with continuous/long term treatment with 0.2 M 9cis-RA and 0.5 M Kenpaullone starting on day 14 of the protocol (i.e. on day 1 of the maturation step) and received thin Fibronectin-Collagen I-overlays on day 14 and 16 of the protocol (i.e. on day 1 and 3 of the differentiation and maturation phase) and is refreshed thereafter once a week on day 23, 30, 37 and so on (i.e. on day 9, 16, 23 and so on of the differentiation and maturation phase).
(263) The thin Fibronectin-Collagen I-overlay is applied as following: Prepare a 3 mg/ml rat tail Collagen I-solution by diluting the Collagen I stock with 0.02M acetic acid. Pre-warm the cell culture medium to room temperature and add 8 l of the 3 mg/ml Collagen I-solution per ml medium (=25 g Collagen I/ml) and 50 l of a 1 mg/ml Fibronectin solution per ml medium (=50 g Fibronectin/ml). Remove the old medium from the cultures and add 0.5 ml of the Collagen I and Fibronectin-containing medium per cm.sup.2 culture surface (=12.5 g Collagen I/cm.sup.2 and 25 g Fibronectin/cm.sup.2). For refreshing the overlay once a week, add 4 l of the 3 mg/ml Collagen I-solution per ml medium (=6.25 g/ml) and 10 l of a 1 mg/ml Fibronectin solution per ml medium (=5 g/ml).
(264) For analysis of mRNA expression (
(265) For analysing CYP enzyme activity in hESC- and hiPSC-derived hepatocytes on day 22, 29 and 36 (i.e. on day 8, 15 and 22 of the maturation step) and human primary hepatocytes 4, 24, 48, 72 and 96 hr after plating, cell cultures were subjected to a CYP activity assay according to the following protocol: Cells are washed twice with warm Williams medium E w/o phenol red (+0.1% PEST). Then CYP activity assay, consisting of warm Williams medium E w/o phenol red (+0.1% PEST), 2 mM L-Glutamine, 25 mM HEPES, 26 M Phenacetin (model substrate for CYP1A), 9 M Diclofenac (model substrate for CYP2C9) and 3 M Midazolam (model substrate for CYP3A), is added to the cells (e.g. 220 l/24 well) and incubated for 16 hr at 37 C. Then supernatant is collected and centrifuged for 20 min at 10.000 rcf at 4 C. Subsequently, 120 l of the supernatant is transferred into a 96 well plate which is sealed with a tight seal tape and stored at 20 or 80 C. until LC/MS-analysis of metabolite formation: Acetaminophen (Paracetamol) for CYP1A, OH-Diclofenac for CYP2C9 and OH-Midazolam for CYP3A,
(266) Results:
(267) The inventors have further investigated the long-term effects of early DNA-demethylation treatment combined with late RA+matrix overlay+Kenpaullone to determine whether hepatic phenotype and expression of hepatic markers genes is maintained after long periods in culture.
(268)
Example 10
Validation of Improved Definitive Endoderm Phenotype in hESC- and hiPSC-Derived DE Treated with a DNA Demethylation Agent
(269) Procedure:
(270) Following the basic protocol C (both for hESC- and hiPSC-derived hepatocytes), cells were treated with 10 nM 5-aza-2-deoxycytidine at different time points and for different durations during the pre-endodermal phase, e.g. on day 2-3, 2-4, 3-4 and 4-6 of the protocol (hESC-DE: no 5azadC n=4, 5azadC d2-3 n=4, d3-4 n=1, d2-4 n=1, d4-6 n=1; hiPSC-DE: no 5azadC n=5, 5azadC d2-3 n=5, d3-4 n=2, d2-4 n=1, d4-6 n=1; with n being the number of individual experiments).
(271) For analysis of mRNA expression, hESC- and hiPSC-derived DE-cells were harvested on day 7 of the protocol and gene expression was analysed using qRT-PCR, normalised to the house-keeping gene CREBBP, and the results presented as relative quantification normalised to a calibrator.
(272) Results:
(273)
(274) DE derived from hESC treated with 10 nM 5-aza-2-deoxycytidine on day 2-3 (
(275)
(276) HiPSC-derived DE treated with 10 nM 5-aza-2-deoxycytidine on day 2-3 (
(277)
(278) HiPSC-derived DE treated with 10 nM 5-aza-2-deoxycytidine on day 2-3 has much less Oct4-immunopositive cells at day 7 compared to untreated controls, i.e. less undifferentiated cells are left and the DE is more homogeneous after treatment with a demethylating agent.
(279)
(280) Expression of the stem cell marker Oct4 is much lower in hESC- and hiPSC-derived DE treated with 10 nM 5azadC on day 2-3, 3-4, 2-4, and 4-6 than in untreated controls (
(281) Taken together, the treatment of the cells with a DNA demethylation agent during the pre-endodermal phase led to improved DE morphology and DE cell yield in both hESC and hiPSC derived cells (
Example 11
Highly Homogeneous Definitive Endoderm Derived from a Panel of 27 hPSC Lines Upon Treatment with a DNA Demethylating Agent During DE Differentiation
(282) Procedure:
(283) Following the basic protocols C or D, cells derived from 27 hPSC lines were treated with nM 5-aza-2-deoxycytidine on day 2-3 during the pre-endodermal phase (protocol C: ChiPSC14, ChiPSC19, ChiPSC22, P11015, SA167, SA181, SA461, and Va19; protocol D: ChiPSC4, ChiPSC6b, ChiPSC7, ChiPSC8, ChiPSC9, ChiPSC10, ChiPSC11, ChiPSC13, ChiPSC15, ChiPSC17, ChiPSC18, ChiPSC19, ChiPSC20, ChiPSC21, ChiPSC23, ChiPSC24, P11012, P11021, P11025, and SAl21). 23 out of 27 hPSC lines were tested with both protocols C and D. Out of these 23 lines, only 4 cell lines (ChiPSC14, ChiPSC23, P11015, and P11032) could only be differentiated with one of the two protocols. Four hPSC lines (ChiPSC8, ChiPSC9, ChiPSC10, and ChiPSC11) were only tested with protocol D.
(284) For analysis of mRNA expression, hESC- and hiPSC-derived DE-cells were harvested on day 7 of the protocol and gene expression was analysed using qRT-PCR, normalised to the house-keeping gene CREBBP, and the results presented as relative quantification normalised to a calibrator.
(285) Results:
(286)
(287) Using the basic protocols C or D including a DNA demethylating treatment on day 2-3 during the pre-endodermal phase, undifferentiated stem cells from 27 different hPSC lines could be differentiated into highly homogeneous DE displaying low mRNA expression levels of the stem cell markers Oct-4 and Nanog (
(288) Taken together, the treatment of the cells during the pre-endodermal phase with a DNA demethylating agent allows derivation of homogeneous DE with low expression levels of stem cell markers and high expression levels of DE markers from all hPSC lines tested. The derivation of homogeneous DE is crucial for derivation of homogeneous hepatocyte cultures which could be obtained from all lines tested (data not shown).
(289) Therefore the skilled person wishing to produce a homogeneous population of definitive endoderm cells (and hepatocytes) from any given hPSC line can include a treatment with a DNA demethylating agent, for instance, on day 2-3 during the pre-endodermal phase.
Example 12
Both DNA Demethylating Agents 5-Aza-2-Deoxycytidine and 5-Azacytidine Improve the Definitive Endoderm Phenotype in hESC- and hiPSC-Derived DE
(290) Procedure:
(291) Following the basic protocols C (P11032, SA181) or D (P11012), cells derived from 3 different hPSC lines were treated with either 10 nM 5-aza-2-deoxycytidine or 1 M 5-azacytidine on day 2-3 during the pre-endodermal phase.
(292) For analysis of mRNA expression, hESC- and hiPSC-derived DE-cells were harvested on day 7 of the protocol and gene expression was analysed using qRT-PCR, normalised to the house-keeping gene CREBBP, and the results presented as relative quantification normalised to a calibrator.
(293) Results:
(294)
(295) A, B) Without treatment with a demethylating agent, the three hPSC lines P11032, SA181 and P11012 produced heterogeneous DE with relatively high mRNA expression of stem cell markers Oct4 and Nanog (
(296) C, D) No significant changes in mRNA expression of the DE markers Sox17 and Cxcr4 could be observed upon treatment with 10 nM 5-aza-2-deoxycytidine or 1 M 5-azacytidine (
(297) Taken together, treatment with both DNA demethylating agents 5-aza-2-deoxycytidine (5azadC) and 5-azacytidine (5azaC) allows derivation of homogeneous DE from hPSC lines, giving otherwise heterogeneous DE if untreated.
(298) Therefore the skilled person wishing to produce a homogeneous population of definitive endoderm cells can select from one or more DNA-demethylation agents and employ them e.g. at days 2-3 during differentiation of pluripotent stem cell types.
Example 13
Further Improvement of Functionality in Hepatocyte-Like Cells Derived from hESC and hiPSC Treated with a Demethylating Agent, Two Activators of a Retinoic Acid Responsive Receptor, Kenpaullone and a Matrix Overlay
(299) Procedure:
(300) Following the basic protocol D (hiPSC-derived hepatocytes), differentiating hPS cells in the pre-endodermal phase were treated with 10 nM of the DNA demethylating agent 5-aza-2-deoxycytidine for 24 hr on day 2 to 3 of the protocol. Later in the protocol, hPS cell derived hepatic progenitor cells and hepatocyte-like cells cultured on a Fibronectin-based coating were treated with continuous/long term treatment with 0.2 M 9cis-RA and 0.5 M Kenpaullone starting on day 14 of the protocol (i.e. on day 1 of the differentiation and maturation phase) and received thin Fibronectin-Collagen I-overlays on day 14 and 16 of the protocol (i.e. on day 1 and 3 of the differentiation and maturation phase) and which are refreshed thereafter once a week on day 23, 30, 37 and so on (i.e. on day 9, 16, 23 and so on of the differentiation and maturation phase). One group received in addition to the described treatment 0.2 M 13cis-RA starting from day 21 of the protocol (i.e. on day 7 of the differentiation and maturation phase).
(301) The thin Fibronectin-Collagen I-overlay is applied as following: Prepare a 3 mg/ml rat tail Collagen I-solution by diluting the Collagen I stock with 0.02M acetic acid. Pre-warm the cell culture medium to room temperature and add 8 l of the 3 mg/ml Collagen I-solution per ml medium (=25 g Collagen I/ml) and 50 l of a 1 mg/ml Fibronectin solution per ml medium (=50 g Fibronectin/ml). Remove the old medium from the cultures and add 0.5 ml of the Collagen I and Fibronectin-containing medium per cm.sup.2 culture surface (=12.5 g Collagen I/cm.sup.2 and 25 g Fibronectin/cm.sup.2). For refreshing the overlay once a week, add 4 l of the 3 mg/ml Collagen I-solution per ml medium (=6.25 g/ml) and 10 l of a 1 mg/ml Fibronectin solution per ml medium (=5 g/ml).
(302) For analysis of mRNA expression, hESC- and hiPSC-derived hepatocytes were harvested on day 36 of the protocol (i.e. on day 22 of the differentiation and maturation phase) and human primary hepatocytes 48 hr after plating. Gene expression was analysed using qRT-PCR, normalised to the house-keeping gene CREBBP, and the results presented as relative quantification normalised to a calibrator.
(303) For analysing CYP enzyme activity in hiPSC-derived hepatocytes on day 36 (i.e. on day 22 of the differentiation and maturation phase), HepG2 and human primary hepatocytes 48 hr after plating, cell cultures were subjected to a CYP activity assay according to the following protocol: Cells are washed twice with warm Williams medium E w/o phenol red (+0.1% PEST). Then CYP activity assay, consisting of warm Williams medium E w/o phenol red (+0.1% PEST), 2 mM L-Glutamine, 25 mM HEPES, 10 M Phenacetin (model substrate for CYP1A), 10 M Bupropion (model substrate for CYP2B6), 10 M Diclofenac (model substrate for CYP2C9), 10 M Bufuralol (model substrate for CYP2D6) and 5 M Midazolam (model substrate for CYP3A), is added to the cells (e.g. 220 l/24 well) and incubated for 16 hr at 37 C. Then supernatant is collected and centrifuged for 20 min at 10.000 rcf at 4 C. Subsequently, 120 l of the supernatant is transferred into a 96 well plate which is sealed with a tight seal tape and stored at 20 or 80 C. until LC/MS-analysis of metabolite formation: Acetaminophen (Paracetamol) for CYP1A, OH-Bupropion for CYP2B6, OH-Diclofenac for CYP2C9, OH-Bufuralol for CYP2D6, and OH-Midazolam for CYP3A,
(304) Results:
(305) The inventors have further investigated the effects of treatment with an additional activator of a retinoic acid responsive receptor in addition to early DNA-demethylation treatment and with late RA+matrix overlay+Kenpaullone treatment to determine if this further improved hepatocyte maturation.
(306)
(307) HiPSC-derived hepatocyte-like cells obtained by treatment with a DNA demethylating agent during early endodermal development and exposure to 9cis RA, Kenpaullone and matrix overlay during the differentiation and maturation phase showed a further increase of CYP1A-, CYP2B6-, CYP2C9-, CYP2D6- and CYP3A-activity upon treatment with 13cis RA.
(308)
(309) HiPSC-derived hepatocyte-like cells obtained by treatment with a DNA demethylating agent during early endodermal development and exposure to 9cis RA, Kenpaullone and matrix overlay together with the additional activator of a retinoic acid responsive receptor 13cis RA during the differentiation and maturation phase showed the highest mRNA expression levels of CYP2C9, CYP3A4, CYP3A5 and PXR.
(310) In contrast to an increase of CYP2B6 activity (
(311) Thus the skilled person may employ treatment with more than one activator of a retinoic acid responsive receptor, e.g. with two, three, four or more activators, in order to obtain hepatocyte-like cells with the most mature characteristics.
Example 14
Further Improvement of Functionality in Hepatocyte-Like Cells Derived from hESC and hiPSC Treated with a Demethylating Agent, Retinoic Acid, Kenpaullone and More Complex Matrix Overlay
(312) Procedure:
(313) Following the basic protocols C (hESC-derived hepatocytes) and D (hiPSC-derived hepatocytes), cells in the pre-endodermal phase were treated with 10 nM of the DNA demethylating agent 5-aza-2-deoxycytidine for 24 hr on day 2 to 3 of the protocol.
(314) Later in the protocol, hPS cell derived hepatic progenitor cells and hepatocyte-like cells derived from hPS cells were treated with continuous/long term treatment with 0.2 M 9cis-RA and 0.5 M Kenpaullone starting on day 14 of the protocol (i.e. on day 1 of the differentiation and maturation phase) and received thin Fibronectin-Collagen I-overlays on day 14 and 16 of the protocol (i.e. on day 1 and 3 of the differentiation and maturation phase) and which are refreshed thereafter once a week on day 23, 30, 37 and so on (i.e. on day 9, 16, 23 and so on of the differentiation and maturation phase).
(315) One experimental group was grown on a liver matrix like-coating, consisting of Fibronectin, Collagen I, Collagen IV, Laminin, Nidogen/Entactin and Biglycan, and received a thin liver matrix like-overlay consisting of Fibronectin, Collagen I, Collagen IV, Laminin, Nidogen/Entactin and Biglycan.
(316) Another experimental group was cultured on a Fibronectin-Collagen I-basal lamina mix-coating, consisting of Fibronectin, Collagen I and a preparation of human extracellular matrix (including collagens, laminin, fibronectin, tenascin, elastin, proteoglycans and glycosaminoglycans), and received a thin Fibronectin-Collagen I-basal lamina mix-overlay, consisting of Fibronectin, Collagen I and a preparation of human extracellular matrix (including collagens, laminin, fibronectin, tenascin, elastin, proteoglycans and glycosaminoglycans).
(317) The control group was grown on the standard Fibronectin-based coating and received thin Fibronectin-Collagen I-overlays on day 14 and 16 of the protocol (i.e. on day 1 and 3 of the differentiation and maturation phase) and which are refreshed thereafter once a week on day 23, 30, 37 and so on (i.e. on day 9, 16, 23 and so on of the differentiation and maturation phase).
(318) The thin Fibronectin-Collagen I-overlay is applied as following: Prepare a 3 mg/ml rat tail Collagen I-solution by diluting the Collagen I stock with 0.02M acetic acid. Pre-warm the cell culture medium to room temperature and add 8 l of the 3 mg/ml Collagen I-solution per ml medium (=25 g Collagen I/ml) and 50 l of a 1 mg/ml Fibronectin solution per ml medium (=50 g Fibronectin/ml). Remove the old medium from the cultures and add 0.5 ml of the Collagen I and Fibronectin-containing medium per cm.sup.2 culture surface (=12.5 g Collagen I/cm.sup.2 and 25 g Fibronectin/cm.sup.2). For refreshing the overlay once a week, add 4 l of the 3 mg/ml Collagen I-solution per ml medium (=6.25 g/ml) and 10 l of a 1 mg/ml Fibronectin solution per ml medium (=5 g/ml).
(319) For the thin liver matrix like-overlay, add 8 l of the 3 mg/ml Collagen I-solution per ml medium (=25 g Collagen I/ml), 50 l of a 1 mg/ml Fibronectin solution per ml medium (=50 g Fibronectin/ml), 1.2 l of a 0.5 mg/ml Collagen IV-solution per ml medium (=0.6 g/ml), 6 l of a 100 g/ml Nidogen/Entactin-solution per ml medium (=0.6 g/ml), 6 l of a 100 g/ml Laminin1-solution per ml medium (=0.6 g/ml), and 6 l of a 200 g/ml Biglycan-solution per ml medium (=1.2 g/ml). For the thin Fibronectin-Collagen I-basal lamina-overlay, add 8 l of the 3 mg/ml Collagen I-solution per ml medium (=25 g Collagen I/ml), 50 l of a 1 mg/ml Fibronectin solution per ml medium (=50 g Fibronectin/ml), and 6 l of a 1 mg/ml human extracellular matrix-preparation per ml medium (=6 g/ml), An example for a suitable human extracellular matrix preparation is MaxGel from Sigma-Aldrich.
(320) For analysing CYP enzyme activity in hESC- and hiPSC-derived hepatocytes on day 36 (i.e. on day 22 of the differentiation and maturation phase), HepG2 and human primary hepatocytes 48 hr after plating, cell cultures were subjected to a CYP activity assay according to the following protocol: Cells are washed twice with warm Williams medium E w/o phenol red (+0.1% PEST). Then CYP activity assay, consisting of warm Williams medium E w/o phenol red (+0.1% PEST), 2 mM L-Glutamine, 25 mM HEPES, 10 M Phenacetin (model substrate for CYP1A), 10 M Bupropion (model substrate for CYP2B6), 10 M Diclofenac (model substrate for CYP2C9), 10 M Bufuralol (model substrate for CYP2D6) and 5 M Midazolam (model substrate for CYP3A), is added to the cells (e.g. 220 l/24 well) and incubated for 16 hr at 37 C. Then supernatant is collected and centrifuged for 20 min at 10.000 rcf at 4 C. Subsequently, 120 l of the supernatant is transferred into a 96 well plate which is sealed with a tight seal tape and stored at 20 or 80 C. until LC/MS-analysis of metabolite formation: Acetaminophen (Paracetamol) for CYP1A, OH-Bupropion for CYP2B6, OH-Diclofenac for CYP2C9, OH-Bufuralol for CYP2D6, and OH-Midazolam for CYP3A,
(321) Results:
(322) The inventors have further investigated the effects of treatment with more complex, ECM-like coatings and overlays in addition to early DNA-demethylation treatment and late retinoic acid and Kenpaullone treatment to determine if this further improved hepatocyte maturation compared to the standard Fibronectin-based coating and the thin Fibronectin-Collagen I-overlay.
(323)
(324) HESC- and hiPSC-derived hepatocyte-like cells obtained by treatment with a DNA demethylating agent during early endodermal development and exposure to retinoic acid, Kenpaullone and a matrix overlay during the differentiation and maturation phase showed a higher CYP2C9-, and CYP3A-activity when both coating and overlay were more complex and ECM-like compared to the standard Fibronectin-based coating and the thin Fibronectin-Collagen I-overlay of the control group.
(325) Thus the skilled person may employ treatment with more complex coatings and overlays resembling the liver matrix in order to obtain hepatocyte-like cells with the most mature characteristics.
Example 15
Improvement of Functionality in Hepatocyte-Like Cells Derived from hiPSC Treated with a Demethylating Agent, Kenpaullone and 9 Cis Retinoic Acid or Analogs of 9 Cis Retinoic Acid
(326) Procedure:
(327) Following the basic protocol D, cells in the pre-endodermal phase were treated with 10 nM of the DNA demethylating agent 5-aza-2-deoxycytidine for 24 hr on day 2 to 3 of the protocol.
(328) Later in the protocol, hPS cell derived hepatic progenitor cells and hepatocyte-like cells derived from hPS cells were treated with continuous/long term treatment with 0.2 M 9cis-RA and 0.5 M Kenpaullone starting on day 14 of the protocol (i.e. on day 1 of the differentiation and maturation phase) and received thin Fibronectin-Collagen I-overlays on day 14 and 16 of the protocol (i.e. on day 1 and 3 of the differentiation and maturation phase) and which are refreshed thereafter once a week on day 23, 30, 37 and so on (i.e. on day 9, 16, 23 and so on of the differentiation and maturation phase).
(329) Some experimental groups were treated with 0.5 M all trans-retinoic acid (ATRA), 5 M AM580, 0.2 M 13cis-RA, 5 M LGD1069, 5 M LG100268 or 5 M SR11237 instead of 0.2 M 9cis-RA.
(330) The thin Fibronectin-Collagen I-overlay is applied as following: Prepare a 3 mg/ml rat tail Collagen I-solution by diluting the Collagen I stock with 0.02M acetic acid. Pre-warm the cell culture medium to room temperature and add 8 l of the 3 mg/ml Collagen I-solution per ml medium (=25 g Collagen I/ml) and 50 l of a 1 mg/ml Fibronectin solution per ml medium (=50 g Fibronectin/ml). Remove the old medium from the cultures and add 0.5 ml of the Collagen I and Fibronectin-containing medium per cm.sup.2 culture surface (=12.5 g Collagen I/cm.sup.2 and 25 g Fibronectin/cm.sup.2). For refreshing the overlay once a week, add 4 l of the 3 mg/ml Collagen I-solution per ml medium (=6.25 g/ml) and 10 l of a 1 mg/ml Fibronectin solution per ml medium (=5 g/ml).
(331) For analysing CYP enzyme activity in hESC-derived hepatocytes on day 36 (i.e. on day 22 of the differentiation and maturation phase), cell cultures were subjected to a CYP activity assay according to the following protocol: Cells are washed twice with warm Williams medium E w/o phenol red (+0.1% PEST). Then CYP activity assay, consisting of warm Williams medium E w/o phenol red (+0.1% PEST), 2 mM L-Glutamine, 25 mM HEPES, 10 M Phenacetin (model substrate for CYP1A), 10 M Bupropion (model substrate for CYP2B6), 10 M Diclofenac (model substrate for CYP2C9), 10 M Bufuralol (model substrate for CYP2D6) and 5 M Midazolam (model substrate for CYP3A), is added to the cells (e.g. 220 l/24 well) and incubated for 16 hr at 37 C. Then supernatant is collected and centrifuged for 20 min at 10.000 rcf at 4 C. Subsequently, 120 l of the supernatant is transferred into a 96 well plate which is sealed with a tight seal tape and stored at 20 or 80 C. until LC/MS-analysis of metabolite formation: Acetaminophen (Paracetamol) for CYP1A, OH-Bupropion for CYP2B6, OH-Diclofenac for CYP2C9, OH-Bufuralol for CYP2D6, and OH-Midazolam for CYP3A,
(332) Results:
(333) The inventors have investigated if other RXR- and RAR-agonist besides 9cis-RA can induce an improved functionality of hiPSC-derived hepatocytes.
(334)
(335) Treatment with 13cis-RA increases CYP2C9 activity to similar levels as upon treatment with 9cis-RA whereas treatment with SR11237, ATRA, AM580, LGD1069 and LG100268 leads to a slightly smaller increase (
(336) Thus the skilled person may use other RXR- and RAR-agonist, e.g. 13cis-RA, ATRA, AM580, LGD1069, LG100268 and SR11237, besides 9cis-RA for obtaining more mature hepatocyte-like cells.
Example 16
Improvement of Functionality in Hepatocyte-Like Cells Derived from hESC and hiPSC Treated with a Demethylating Agent, 9 Cis Retinoic and Kenpaullone Acid or Analogs of Kenpaullone
(337) Procedure:
(338) Following the basic protocols C (hESC-derived hepatocytes) and D (hiPSC-derived hepatocytes), cells in the pre-endodermal phase were treated with 10 nM of the DNA demethylating agent 5-aza-2-deoxycytidine for 24 hr on day 2 to 3 of the protocol. Later in the protocol, hPS cell derived hepatic progenitor cells and hepatocyte-like cells derived from hPS cells were treated with continuous/long term treatment with 0.2 M 9cis-RA and 0.5 M Kenpaullone starting on day 14 of the protocol (i.e. on day 1 of the differentiation and maturation phase) and received thin Fibronectin-Collagen I-overlays on day 14 and 16 of the protocol (i.e. on day 1 and 3 of the differentiation and maturation phase) and which are refreshed thereafter once a week on day 23, 30, 37 and so on (i.e. on day 9, 16, 23 and so on of the differentiation and maturation phase).
(339) One experimental group was treated with 0.5 M Indirubin-3-oxime instead of 0.5 M Kenpaullone.
(340) The thin Fibronectin-Collagen I-overlay is applied as following: Prepare a 3 mg/ml rat tail Collagen I-solution by diluting the Collagen I stock with 0.02M acetic acid. Pre-warm the cell culture medium to room temperature and add 8 l of the 3 mg/ml Collagen I-solution per ml medium (=25 g Collagen I/ml) and 50 l of a 1 mg/ml Fibronectin solution per ml medium (=50 g Fibronectin/ml). Remove the old medium from the cultures and add 0.5 ml of the Collagen I and Fibronectin-containing medium per cm.sup.2 culture surface (=12.5 g Collagen I/cm.sup.2 and 25 g Fibronectin/cm.sup.2). For refreshing the overlay once a week, add 4 l of the 3 mg/ml Collagen I-solution per ml medium (=6.25 g/ml) and 10 l of a 1 mg/ml Fibronectin solution per ml medium (=5 g/ml).
(341) For analysing CYP enzyme activity in hESC- and hiPSC-derived hepatocytes on day 36 (i.e. on day 22 of the differentiation and maturation phase), cell cultures were subjected to a CYP activity assay according to the following protocol: Cells are washed twice with warm Williams medium E w/o phenol red (+0.1% PEST). Then CYP activity assay, consisting of warm Williams medium E w/o phenol red (+0.1% PEST), 2 mM L-Glutamine, mM HEPES, 10 M Phenacetin (model substrate for CYP1A), 10 M Bupropion (model substrate for CYP2B6), 10 M Diclofenac (model substrate for CYP2C9), 10 M Bufuralol (model substrate for CYP2D6) and 5 M Midazolam (model substrate for CYP3A), is added to the cells (e.g. 220 l/24 well) and incubated for 16 hr at 37 C. Then supernatant is collected and centrifuged for 20 min at 10.000 rcf at 4 C. Subsequently, 120 l of the supernatant is transferred into a 96 well plate which is sealed with a tight seal tape and stored at 20 or 80 C. until LC/MS-analysis of metabolite formation: Acetaminophen (Paracetamol) for CYP1A, OH-Bupropion for CYP2B6, OH-Diclofenac for CYP2C9, OH-Bufuralol for CYP2D6, and OH-Midazolam for CYP3A,
(342) Results:
(343) The inventors have investigated if other CDK- and GSK3-inhibitors besides Kenpaullone can induce an improved functionality of hiPSC-derived hepatocytes.
(344)
(345) Treatment with Indirubin-3-oxime increases CYP2C9 and 3A activity to similar levels as upon treatment with Kenpaullone both in hiPSC-derived hepatocytes (
(346) Thus the skilled person may use other CDK- and GSK3-inhibitors besides Kenpaullone for obtaining more mature hepatocyte-like cells.
REFERENCES
(347) Allenby, G. et al (1993) Retinoic acid receptors and retinoid X receptors: Interactions with endogenous retinoic acids. PNAS Biochemistry, 90:30-34 Brolen, G. et al. (2010) Hepatocyte-like cells derived from human embryonic stem cells specifically via definitive endoderm and a progenitor stage. J Biotechnol. 1; 145(3):284-94 Chen, Y. F. et al. (2012) Rapid generation of mature hepatocyte-like cells from human induced pluripotent stem cells by an efficient three-step protocol. Hepatology. 2012 55(4):1193-203 Chung, Y. et al. (2008) Human Embryonic Stem Cell Lines Generated without Embryo Destruction. doi: 10.1016/j.stem.2007.12.013 Duan, Y. et al. Differentiation and characterization of metabolically functioning hepatocytes from human embryonic stem cells. Stem Cells. 28(4):674-86 Dunn, J et al. (1991) Long-term in Vitro function of adult hepatocytes in a collagen sandwich configuration. Biotechnol. Prog. 7:237-245 Funakoshi, N. et al. (2011) Comparison of hepatic-like cell production from human embryonic stem cells and adult liver progenitor cells: CAR transduction activates a battery of detoxification genes. Stem Cell Rev. 7(3):518-31 Hatzis, P. et al (2001) Regulatory Mechanisms Controlling Human Hepatocyte Nuclear Factor 4a Gene Expression. Mol. Cell. Biol. November 2001:7320-7330 Hay, D. et al (2007) Direct differentiation of human embryonic stem cells to hepatocyte-like cells exhibiting functional activities. Cloning Stem Cells. 2007 Spring; 9(1):51-62. Erratum in: Cloning Stem Cells. 2009 March; 11(1):209. Hay, D. et al (2008) Efficient differentiation of hepatocytes from human embryonic stem cells exhibiting markers recapitulating liver development in vivo. Stem Cells. April; 26(4):894-902. Heins, N. et al (2004) Derivation, characterization, and differentiation of human embryonic stem cells. Stem Cells. 22(3):367-76. Heyman, R A. et al. (1992) 9-cis retinoic acid is a high affinity ligand for the retinoid X receptor. Cell. 1992 68(2):397-406. Idres, N. et al (2002) Activation of retinoic acid receptor-dependent transcription by all-trans-retinoic acid metabolites and isomers. J Biol Chem. 277(35):31491-8. Klimanskaya, I. et al (2006) Human embryonic stem cell lines derived from single blastomeres. Nature, November 23; 444(7118):481-5. Epub 2006 Aug. 23. Erratum in: Nature. 2006 Nov. 23; 444(7118):512. Nature. 2007 Mar. 15; 446(7133):342. Magee, T. et al (1998) Retinoic Acid Mediates Down-regulation of the -Fetoprotein Gene through Decreased Expression of Hepatocyte Nuclear Factors. J. Bio. Chem. 273, 45:30024-30032 Martin M. et al (2005) Human embryonic stem cells express an immunogenic nonhuman sialic acid. Nat Med. February; 11(2):228-32. Mfopou, J. et al. (2010) Noggin, Retinoids, and Fibroblast Growth Factor Regulate Hepatic or Pancreatic Fate of Human Embryonic Stem Cells. Gastroenterology 138:2233-2245. Mercader, A. et al (2009) Human Embryo Culture. Essential Stem Cell Methods, Chapter 16, Academic Press, 1.sup.st Edition, Eds. Lanza, R. and Klimanskaya, I. Page, J et al. (2007) Gene expression profiling of extracellular matrix as an effector of human hepatocyte phenotype in primary cell culture. Tox. Sci. 97(2):384-397 Qian, A. et al. (2000) Identification of Retinoic Acid-Responsive Elements on the HNF1a and HNF4 Genes. Biochem. Biophys. Res. Comm. 276:837-842 Si-Tayeb, K. et al. (2010) Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells Hepatology. 51(1):297-305. Song. Z. et al. (2009) Efficient generation of hepatocyte-like cells from human induced pluripotent stem cells. Cell Res. 19(11):1233-42 Sullivan, G. J. et al. (2010) Generation of functional human hepatic endoderm from human induced pluripotent stem cells. Hepatology. 51(1):329-35. Takahashi, K. et al (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell November 30; 131(5):861-72. Thomson, J. et al. (1998) Embryonic stem cell lines derived from human blastocysts. Science. November 6; 282(5391):1145-7. Erratum in: Science 1998 Dec. 4; 282(5395):1827. Touboul, T. et al. (2010) Generation of Functional Hepatocytes from Human Embryonic Stem Cells Under Chemically Defined Conditions that Recapitulate Liver Development. Hepatology 51:1754-1765 Turner, R. et al. (2011) Human hepatic stem cell and maturational liver lineage biology. Hepatology. 53(3):1035-45 Wang, Y. et al. (2011) Lineage restriction of human hepatic stem cells to mature fates is made efficient by tissue-specific biomatrix scaffolds. Hepatology. 53(1):293-305. Yu, J. and Thomson, J. (2009) Induced Puripotent Stem Cell Derivation. Essentials of Stem Cell Biology, Chapter 37, Academic Press, 2.sup.nd Edition (2009), Eds. Lanza, R. et al. Zhou H. et al (2009). Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell. 4(5):381-4.