Directed differentiation and maturation of pluripotent cells into hepatocyte like cells by modulation of Wnt-signalling pathway

Abstract

Provided are improved methods using Glycogen synthase kinase 3 (GSK3) inhibitors by which endodermal cells, notably endodermal cells derived from human pluripotent stem cells (hPS), such as but not limited to hiPS-cells and hES-cells may be differentiated into hepatocyte like cells. The specific modulation of wingless integration gene (WNT)-signalling pathway and use of GSK3 inhibitors achieve direct differentiation and maturation of hepatocytes derived from human pluripotent stem (hPS) cells. GSK-3 inhibitors, when added to the growth medium at certain developmental stages, leads to more mature and functional features for the hepatocyte like cells as well as more pure and homogenous populations of hepatocyte like cells. Provided are also hepatocyte like cells obtained by these methods as well as compositions comprising them.

Claims

1. A composition comprising: an in vitro derived hepatocyte-like cell in a media comprising one or more GSK3 inhibitors, wherein said in vitro derived hepatocyte-like cell has elevated expression of the genes CYP1A1, CYP1A2, CYP3A4, CYP7A1, and MRP2 compared to a comparable hepatocyte-like cell obtained under conditions without use of any GSK3 inhibitor.

2. The composition of claim 1, wherein said in vitro derived hepatocyte-like cell has enzymatic activities exceeding a fold change of at least 10 in cytochrome P450 activity when compared to saki comparable hepatocyte-like cell obtained under conditions without use of any GSK3 inhibitor.

3. The composition of claim 1, wherein said in vitro derived hepatocyte-like cell has enzymatic activities exceeding a fold change of at least 13 in cytochrome P450 activity when compared to saki comparable hepatocyte-like cell obtained under conditions without use of any GSK3 inhibitor.

4. The composition of claim 1, wherein said one or more GSK3 inhibitors is selected from the group consisting of: BIO, kenpaullone, SB2167763, Indirubin-3-monoxime, and any combination thereof.

5. The composition of claim 1, wherein said one or more GSK3 inhibitors is BIO.

6. The composition of claim 1, wherein said one or more GSK3 inhibitors is kenpaullone.

7. The composition of claim 1, wherein said one or more GSK3 inhibitors is SB2167763.

8. The composition of claim 1, wherein said one or more GSK3 inhibitors is Indirubin-3-monoxime.

9. The composition of claim 1, wherein said in vitro derived hepatocyte-like cell further has elevated expression of the gene CYP2C9 compared to the comparable hepatocyte-like cell obtained under conditions without use of any GSK3 inhibitor.

10. The composition of claim 1, wherein said in vitro derived hepatocyte-like cell further has elevated expression of the genes OATP2 and AAT compared to the comparable hepatocyte-like cell obtained under conditions without use of any GSK3 inhibitor.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1. Overview of the derivation protocol of hepatocyte-like cells from hPS, as further outlined in example 2 and 3. The striped bar describes the times for addition of a GSK3 inhibitor.

(2) FIG. 2. Detailed variants of the protocols for differentiating hPS towards hepatocyte-like cells, protocol i to iv, as further described in examples 4-11.

(3) 2 i. shows the control culture conditions without GSK-3 inhibitor added.

(4) 2 ii. shows one aspect of the invention, in which the GSK-3 inhibitor is added to the growth medium after initial differentiation, hence when the cells are showing characteristics similar to type of the endodermal or more specifically definitive endoderm lineage. In this aspect, the GSK-3 inhibitor is removed when the cells are showing characteristic similar to hepatic progenitor cells.

(5) 2 iii. shows one aspect of the invention, in which the GSK-3 inhibitor is added to the growth medium after differentiation into hepatocyte progenitors. Thus in this aspect of the invention, the GSK-3 inhibitor is added when the cells are showing characteristic similar to hepatic progenitor cells.

(6) 2 iv. shows one aspect of the invention, in which the GSK-3 inhibitor is added to the growth medium after initial differentiation when the cells are showing characteristics similar to type of the endodermal or more specifically definitive endoderm lineage. In one further aspect according to iv. the type and concentration of the GSK-3 inhibitor may be changed during the culturing period.

(7) FIG. 3. Further variants of protocols for differentiating hPS towards hepatocyte-like cells showing media and stages in the protocol of inducing hPS to hepatocyte-like cells. A) shows the control culture conditions without a GSK inhibitor. B) Shows the addition of a GSK3 inhibitor at day 14, or when the cells resemble cell of the hepatic progenitor type. C) Shows the use of a split media (SM) before the addition of the GSK3 inhibitor. The protocols are further described in examples 12-14.

(8) FIGS. 4A-C. Feeder free cultured hPS cells with and without the GSK3 inhibitor, as well as an alternative method where a split media as described in FIG. 3C were compared for their hepatic profile and homogeneity.

(9) A) Results from Activity assay of CYP1A and CYP3A measured by conversion of paracetamol and OH_midazolam respectively. The use of a split media shows that high levels of CYP3AX can be maintained when a GSK3 inhibitor and a split media is used.

(10) B) Shows a possible increase in proliferation by addition of a GSK-3 inhibitor, measured by an increase of the progenitor marker CD44 when a GSK3 inhibitor was added. The split media (SM) gave the same levels as without split media. Two controls were used undifferentiated cells cultured feeder free (UD feeder free) and a perpetual cell line from a hepatocellular carcinoma (HepG2). The levels of AFP, KRT18 (Keratin 18) and KRT19 (Keratin 19) indicate that the cells are maintained in a stage where they still have the ability to proliferate. The cells were 23 days when they were analyzed, n=3.

(11) C) Immunolocalisation of Beta-catenin and Dapi staining of hES-HEP-BIO (A) and hES-HEP+BIO (B) Beta-catenin is localized at the cell membrane in hES-HEP cultures not treated and treated with BIO. Beta catenin in the cytoplasm and nuclei is commonly observed in BIO treated hES-HEP cultures. Day 21, 20

(12) FIGS. 5A and B. hiPS cells cultured with and without the GSK3 inhibitor were compared for their hepatic profile and homogeneity. Where a GSK3 inhibitor was added it was added at a later stage of the differentiation, according to FIG. 3. Derivation of hepatocytes from human induced pluripotent stem cells (hiPS).

(13) A) Results from Activity Assay of CYP1A, 3A and 2C. The addition of a GSK3 inhibitor gave increased activity of CYP1A and CYP3A. The levels of CYP2C are high in relation to what is usually shown for hepatocyte-like cells derived from hPS cells. The cells were 30 days, n=8. hiPS vs hiPS+BIO.

(14) B) Results of hepatic markers from Q-PCR of the same cells that were analysed by Activity assay (Q-PCR hiPS+/BIO) n=4. The results show a clear increase of CYP1A1, CYP1A2, CYP3A4 and CYP7A1. The levels of CYP2C9 where maintained high for the hiPS cells when a GSK inhibitor was added. The decreased AFP levels indicate maturation. Albumin was maintained at a level high compared to known protocols for the derivation of hepatocyte-like cells from hPS cells. AAT was maintained on a high level. Important transporters such as MRP2, Oct-1, GSTA1, BCEP and OATP2 where maintained at levels significantly higher than HepG2 showing a maturation of the hiPS cell derived hepatocyte-like cells.

(15) FIGS. 6A and B. hES cells cultured with and without the GSK3 inhibitor were compared for their hepatic profile and homogeneity, as described in examples 12 and 13. Where a GSK3 inhibitor was added it was added at a later stage of the differentiation, according to FIG. 3B.

(16) A) Results from Activity Assay of CYP1A, 3A and 2C. The addition of a GSK3 inhibitor gave increased activity of CYP1A, 3A and 2C. The cells were 30 days, n=8. hPS vs hPS+BIO.B)

(17) B) Results of hepatic markers from Q-PCR of the same cells that were analysed by Activity assay (Q-PCR hPS+/BIO) n=4. The results show a clear increase of CYP1A1, CYP1A2, CYP3A4, CYP2C9 and CYP7A1. An increase in AAT was shown when BIO was added. Important transporters such as MRP2, Oct-1, GSTA1, BCEP and OATP2 where maintained at levels showing a maturation of the hPS cell derived hepatocyte-like cells.

(18) FIGS. 7A-C. Induction of Cyp1A by GSK3 inhibitor in hESC derived hepatocytes.

(19) A) Shows Cytochrome P450 activity of CYP1A in hESC-HEP differentiated with and without GSK3 inhibitor (Example 4 (MMI-BIO) vs. Example 8 (MMI+BIO) vs. Example 9 (MMII+BIO)). Analysis are performed at day 16-18 (n=7-8), 20-21 (n=5) and 25 (n=2) respectively, meanSD. The tables list the increase of CYP activity as a fold change value.

(20) B) Show gene expression levels of CYP1A1 and CYP1A2 respectively in hepatocyte-like cells differentiated in the presence of GSK3 inhibitor (Example 9) compared to HepG2. Analysis are performed day 16-18 (n=5), 19-21 (n=4) for CYP1A2 and day 16-19 (n=14), 21-23 (n=6) for CYP1A1, meanSD. (Cells obtained as described in example 9 vs. HepG2 cells vs. hPS cells)

(21) C) show immunocytochemistry of CYP1A2 (red) at day 19 in hepatocyte-like cells differentiated in the presence of GSK3 inhibitor (Example 9) . . . .

(22) FIGS. 8A-E. Differentiation of hESC derived hepatocytes in the presence of GSK3 inhibitor from day 3 purifies hepatocytes from other cell types.

(23) A-C show hESC-HEP at day 17, differentiated by protocol i or ii according to FIG. 2. D-E show hESC-HEP at day 15, differentiated by protocol iii or vi according to FIG. 2,

(24) A) 0 M BIO, protocol i.

(25) B) 1 M BIO day 3-9, protocol ii.

(26) C) 5 M BIO day 3-9, protocol ii.

(27) D) 0 M BIO day 3-9, 1.5 M BIO day 10-15 protocol iii,

(28) E) 3.5 M BIO day 3-9, 1.5 M BIO day 10-15 protocol vi.

(29) A and B show hESC-HEP cultures which are over grown by another cell type where as in C the majority of cells in the culture are hepatocyte-like cells. E represent a purer hESC-HEP culture than D. White arrow: hepatocyte-like cells, black arrow: other cell type than hepatocyte-like cells. Scale bar: 100 m.

(30) FIG. 9. Functional CYP1A activity in hES-HEP cultures differentiated in media supplemented with NaB (an Histone deacetylase inhibitor (HDAC) inhibitor) and BIO (a GSK3 inhibitor), compared to cultures without NaB in the maturation media, suggesting HDAC inhibitors, eg NaB, to potentiate Wnt-signalling mediated transcription. Analysis is performed day 24-25, n=1.

(31) FIGS. 10A-D. Gene expression levels of hepatic markers are induced by BIO supplemented at day 3. Qrt-PCR data is presented as fold change gene expression levels of HepG2 except for BSEP, which graph show fold change of calibrator. N=2-3, meanSEM. FIGS. 10A-D corresponds to example 22.

(32) A) Phase I, drug metabolising enzymes: CYP3A4, CYP3A5, and CYP2C9 show increased expression levels at the addition of BIO. CYP3A7 shows no significant increase

(33) B) Phase II, drug metabolising enzymes: GSTA1 and UGT2B7 shows and increased expression when BIO was added.

(34) C) Phase III, transporters MRP2 and BSEP shows both increased expression when BIO was added.

(35) D) General liver markers: A1AT showed a strong increase at day 28. ALB decreased at day 26 indicating maturation of the hepaticyte-like cells. TAT showed higher expression levels than for the cells cultured with BIO than the ones with out BIO for all days.

(36) FIG. 11. Relative expression levels of UGT hepatic metabolic markers in hESC-HEP generated with or without Wnt-signalling modulation by GSK-3 inhibitor BIO during endoderm to hepatocyte-like cell phase. FC=fold change relative expression, control set 30 to 1 for each example. Graphs show expression levels in cells treated with one of three differentiation/maturation protocols (see example 21) and +/GSK-3 inhibitor treatment for activity of UGTs on specific UGT substrates: (UGT1A1,[8-estradiol]); (UGT2B, [8-estradiol]; (UGT1A6, [1-Napthol]), (UGT1A9, [Propofol]), (UGT2B7, [Naloxone]). A non-specific control (Methylumbelliferone) was also included. n=number of experiments averaged to obtain results.

(37) FIGS. 12A and B. Relative expression levels of hepatic markers in hESC-HEP generated with modulation of Wnt-signalling by GSK-3 inhibitors other than BIO during later maturation period (day 10 onwards). Treatments shown include: hESC-HEP (negative control; cells never exposed to GSK-inhibitor during differentiation), BIO, SB216763, Kenpaullone, Indirubin-3-O; also included is a second negative control undifferentiated hESC cells (hESC). FIG. 12A shows expression of Phase I enzymes, FIG. 12B shows expression of Phase II enzymes and hepatic markers. GSK-3 inhibitors were present only during later differentiation (from day 10 onwards); also included is a further negative control graph showing the expression levels of a housekeeping gene (Creb) across the various samples.

(38) FIGS. 13A and B. Relative expression levels of hepatic markers in hESC-HEP generated with modulation of Wnt-signalling by GSK-3 inhibitors other than BIO during both mid (days 3-9) and late stage (days 10-23) differentiation. Treatments shown include: hESC-HEP (negative control; cells never exposed to GSK-inhibitors), BIO, SB216763, Kenpaullone, Indirubin-3-O; also included is a second negative control undifferentiated hESC cells (hESC); also included is a further negative control graph showing the expression levels of a housekeeping gene (Creb) across the various samples. FIG. 13A shows expression of Phase I enzymes, FIG. 13B shows expression of Phase II enzymes and hepatic markers.

(39) FIGS. 14A-C. Relative expression levels of Phase I enzymes (A), Phase II enzymes (B) and General hepatic markers (C) in hiPS-HEP cells generated with modulation of Wnt-signalling by GSK-3 inhibitors other than BIO during later (day 10+) differentiation. Treatments shown include: iPS-HEP (negative control; cells never exposed to GSK-inhibitors), BIO, SB216763, Kenpaullone, Indirubin-3-O; also included is a second negative control undifferentiated hiPS cells (iPS).

(40) FIGS. 15A-C. Relative expression levels of Phase I enzymes (A), Phase II enzymes (B) and General hepatic markers (C) in hiPS-HEP cells generated with modulation of Wnt-signalling by GSK-3 inhibitors other than BIO during both mid (days 3-9) and late stage (days 10-23) differentiation. Treatments shown include: iPS-HEP (negative control; cells never exposed to GSK-inhibitors), BIO, SB216763, Kenpaullone, Indirubin-3-O; also included is a second negative control undifferentiated hiPS cells (iPS).

EXAMPLES

(41) In present invention, several modulators of the Wnt pathway have been tested, including GSK inhibitor BIO (GSK inhibitor IX), Kenpaullone, SB216763 and Indirubin-3-oxime. As well known within the field and discussed in the scientific literature as example (Nejak-Bowen et al 2008), the other GSK-3 inhibitors and other molecules effecting the signalling cascade are suggested to have a similar effect for modulation of Wnt signalling pathway. Examples of general culturing and passaging techniques are disclosed in pending applications PCT/EP2004/005033, PCT/EP02/14895, PCT/EP2005/040582, PCT/EP2006/009697, PCT/EP2007/004940 and PCT/EP208/059491.

(42) As laid out in the examples, the starting material may comprise any pluripotent stem cell derived through an initial differentiation towards a definitive or extraembryonic lineage. The starting material may also be any cell of hepatic progenitor lineage.

Example 1

Starting Material for Hepatocytes Derived from Human Pluripotent Stem Cells Maintained on Feeder Cells

(43) 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) cultured on mEF cells (Heins et al 2004, Stem Cells). The cell lines used for this experiment could be, but is not limited to the hES cell line SA002, SA121 and SA181 (Cellartis AB, Gteborg, Sweden) and they can be propagated as described 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. Along with hPS obtained from hESC, hPS cells invention hPS obtained from hiPS (induced pluripotent stem cells) have been used for the derivation of hepatocytes for the examples of this invention.

Example 2

(44) Derivation of hepatocytes from human pluripotent stem cells using a GSK3 inhibitor. Hepatocytes were derived from both hES cells and human hiPS cells according to the protocol in FIG. 1, this protocol gives an overview of the derivation of human hepatocyte-like cells from human pluripotent stem cells.

(45) Before adding the first medium, ID day 0-2, the cultures were washed thoroughly with PBS, twice. The different mediums were prepared freshly and added day 0 (ID day 0-1), 2 (ID day 2-4), 4 and 7-10 every second or third day (VH1), 10-28 every second or third day (MMI or MM II). Cells are passaged at day 4 and replated 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 200 000 cells/cm.sup.2.

(46) The Initial Differentiation (ID) Step

(47) Day 0-1

(48) RPMI 1640 (+0.1% PEST, +1% Glutamax)

(49) 1B27

(50) 100 ng/ml Activin A

(51) 1 mM NaB

(52) Day 2-3

(53) RPMI 1640 (+0.1% PEST+1% Glutamax)

(54) 1B27

(55) 100 ng/ml Activin A

(56) 0.5 mM NaB

(57) Hepatic Progenitor Step

(58) Day 3

(59) +/3.5 M GSK-3 inhibitor (e.g. BIO)

(60) VH1

(61) Day 4-9

(62) VitroHES

(63) 1% DMSO

(64) +/3.5 M GSK-3 inhibitor (e.g. BIO)

(65) Maturation media (MM) I

(66) Day 10-30

(67) WME+SQ (GA1000)+1% Glutamax+0.1% PEST)

(68) 10 ng/ml OsM

(69) 0.1 M DexM

(70) 2 ng/ml bFGF

(71) 10 ng/ml HGF

(72) 0.5% DMSO

(73) 10 mM Nicotinamide

(74) ITS (10 l/ml)

(75) 3 ng/ml Glucagon

(76) +/1.5 M GSK-3 inhibitor (e.g. BIO)

Example 3

(77) As outlined in Example 2 and in FIG. 1, but with maturation medium II (MM II) replacing maturation medium I (MM I).

(78) Maturation Media (MM)

(79) Day 10-30

(80) WME+SQ (GA1000)+1% Glutamax+0.1% PEST)

(81) 10 ng/ml OsM

(82) 0.1 M DexM

(83) 20 ng/ml HGF

(84) 0.5% DMSO

(85) +/1.5 M GSK-3 inhibitor (e.g. BIO)

Example 4

(86) As Example 2, but without addition of a GSK3 inhibitor. Example 4 is a control protocol in which hESC were differentiated into hepatocyte-like cells in the absence of GSK3 inhibitor.

(87) As outlined in FIG. 2) (i-iv), variants of the overview protocol in FIG. 1 were tested. The differentiation procedure follows three stages, first during the initial differentiation (ID) step, partly differentiated cells resembling endodermal cells are formed (day 0-4), second the partly differentiated cells are differentiated into hepatoblasts/hepatic progenitors (day 4-10) and finally the hepatoblasts are matured into hepatocyte-like cells (day 10-30).

(88) Example 4 was performed as schematically depicted in FIG. 2 i.

Example 5

(89) As example 3, but without addition of a GSK3 inhibitor. Included as a control protocol of which hESCs were differentiated into hepatocyte-like cells in the absence of GSK3 inhibitor.

(90) Example 5 was performed as schematically depicted in FIG. 2 i.

Example 6

(91) Schematically depicted in FIG. 2 ii. As example 2 but with 3.5 M GSK3 inhibitor is added at day 3 to day 10 only.

Example 7

(92) Schematically depicted in FIG. 2 ii. As example 3 but with 3.5 M GSK3 inhibitor is added at day 3 to day 10 only.

Example 8

(93) Schematically depicted in FIG. 2 iii.

(94) As example 2 but with 1.5 M GSK3 inhibitor is added at day 10 to day 30 only.

Example 9

(95) Schematically depicted in FIG. 2 iii.

(96) As example 3 but with 1.5 M GSK3 inhibitor is added at day 10 to day 30 only.

Example 10

(97) Schematically depicted in FIG. 2 iv. As example 2 but with 3.5 M GSK3 inhibitor added at day 3 to day 10 and changed to 1.5 M GSK3 inhibitor at day 10 and throughout the protocol.

Example 11

(98) Schematically depicted in FIG. 2 iv. As example 3 but with 3.5 M GSK3 inhibitor added at day 3 to day 10 and changed to 1.5 M GSK3 inhibitor at day 10 and throughout the protocol.

Example 12

Medium and Supplement Factors for Hepatocyte-Like Cells Derived from Feeder Free hES or hiPS

(99) Wash the cells prior initiation of differentiation of the undifferentiated hPS hepatocyte-like cells. Before adding the first medium, Initial differentiation (ID) step day 0-2, the undifferentiated cultures (UD) in T150 flasks were washed thoroughly with PBS or RPMI640, twice. The different mediums were prepared freshly and added daily at day 0 (ID day 0-1), 2, 3 and 4 (ID day 2-4), 4. Then the cells were passaged at a concentration of approximately 200,000 cells/cm.sup.2 to freshly gelatine- or matrigel coated 24 well plates in VH1 medium. The maturation medium was then changed every second day or third day for day 7-10, and for day 10-28 every second or third day (BM2 or ModII).

(100) Example 12 illustrates the derivation without a GSK3 inhibitor (BIO) used as a control for this invention and was carried out as outlined in FIG. 3 A).

(101) The Initial Differentiation (ID) Step

(102) Day 0-1

(103) RPMI 1640 (+0.1% PEST, +1% Glutamax)

(104) 1B27

(105) 100 ng/ml Activin A

(106) 1 mM NaB

(107) ID

(108) Day 2-7

(109) RPMI 1640 (+0.1% PEST)

(110) 1B27

(111) 100 ng/ml Activin A

(112) 0.5 mM NaB

(113) Day 7 the cells are passaged with TrypLE Select. The cells are incubated for 3-7 minutes at 37 C. Diluted and washed with VH medium, spun at 300 g, 5 min. Thereafter, the cells were seeded onto fresh coated dishes.

(114) VitroHES 1 Step (VH1)

(115) Day 7-14

(116) VitroHES (VH)

(117) 1% DMSO

(118) Maturation Media BM2 (or Alternatively MMII (as Described in Example 2)) Day 14-28

(119) WME+SQ (-GA1000, +1% Glutamax+0.1% PEST)

(120) (10 ng/ml OsM)

(121) 0.1 M DexM

(122) 10 ng/ml HGF

(123) 0.5% DMSO

Example 13

(124) Performed as outlined in FIG. 3 B). As example 7 but with 1.4 M GSK3 inhibitor is added at day 14.

Example 14

(125) Performed as outlined in FIG. 3 C) describes the derivation with the cells cultured in a split medium between day 7-9 and a GSK3 inhibitor introduced at day 14.

(126) The Initial Differentiation (ID) Step

(127) Day 0-1

(128) RPMI 1640 (+0.1% PEST, +1% Glutamax)

(129) 1B27

(130) 100 ng/ml Activin A

(131) 1 mM NaB

(132) ID Day 2-7

(133) RPMI 1640 (+0.1% PEST)

(134) 1B27

(135) 100 ng/ml Activin A

(136) 0.5 mM NaB

(137) Day 7 the cells are passaged with TrypLE Select. Incubated for 4 minutes at 37 C. Diluted and washed with VH4 medium, spun at 300 g, 5 min. Thereafter, the cells were seeded onto fresh coated dishes.

(138) Split Media (SM) Day 7-9

(139) RPMI A (+0.1% PEST+1% Glutamax (10 l/ml)

(140) 100 ng/ml aFGF

(141) 5 ng/ml bFGF

(142) 50 ng/ml BMP2

(143) 200 ng/ml BMP4

(144) 0.2% FBS

(145) VitroHES 1 Step (VH1) Day 9-14

(146) VitroHes

(147) 1% DMSO

(148) Maturation Media BM2 (or Alternatively MMII (as Described in Example 2)) Day 14-28

(149) WME+SQ (-GA1000, +1% Glutamax+0.1% PEST)

(150) (10 ng/ml OsM)

(151) 0.1 M DexM

(152) 2 ng/ml bFGF

(153) 10 ng/ml HGF

(154) 0.5% DMSO

(155) 10 mM nicotineamide

(156) 10 g/ml ITS

(157) 3 ng/ml Glucgon

(158) 1.4 M BIO

Example 15

Induction of Cytochrome P450 1A in mEF-Cultured hESC Derived Hepatocytes by GSK3-Inhibitor

(159) MEF-cultured hESCs (hES cells cultured on feeder cells) were differentiated into hepatocyte-like cells according to examples 4, 8 and 9, thus comparing the absence of BIO, a GSK3-inhibitor (Example 4), to the influence of BIO during the maturation step (day 10-26) in maturation medium I and II (MM I and MM II)(Example 8 and 9 respectively). Before adding the first medium, ID day 0-2, the cultures were washed thoroughly with PBS, twice. The different mediums were prepared freshly and added day 0 (DE day 0-1), 2 (DE day 2-4), 4 and 7-10 every other to third day (VH1), 10-26 every second or third day (MMI or MM II). Detailed information about the composition of the different mediums, see examples 4, 8 and 9. At day four, the cells were passaged to new dishes in order to obtain a confluent layer of cells that subsequently was differentiated into hESC-HEP. Briefly, the cells were detached from the culture unit by incubating the cells in an enzyme solution, Tryple Select, for 5 to 10 min. VitroHes-medium was added to the cultures to stop the effect of the enzyme. The detached cells were transferred to tubes and centrifuged for 5 min at 300 g. The supernatant was discarded and VH1-medium added to the cell pellet, which was subsequently dissociated into single cell suspension. Cells were counted in a Brker chamber and seeded out in 0.1% gelatine coated culture units (e.g. in 24-well plates) at a cell density of 150 to 250K cells per cm.sup.2.

(160) At day 16, 18, 20, 21 and 25 hESC-HEP cultures were analyzed for cytochrome P450 1A activity by incubating the substrate Phenacetine to a final concentration of 26 M in Phenol Red-free Williams Medium E, supplemented with 0.1% Penicilline-Streptomycin 2 mM L-Glutamine and 25 mM Hepes. A volume of 220 l diluted substrates were added per well of a 24-well plate. hESC-HEP cultures with substrates were incubated over night. After 16 h, medium was collected and subsequently, centrifuged at 10 000 g, 4 C. for 20 min. Samples were analysed by Liquid chromatography-mass spectrometry (LC-MS) LCMS for presence of the metabolite Paracetamol, biotransformed by the cytochrome p450 enzymes Cyp1A2, 1A1.

(161) Results

(162) hESC-HEP, matured in maturation medium I supplemented with 1.5 M BIO (MMI+BIO) according to Example 8, were able to metabolize phenacetine into paracetamol to a greater extent than the control cultures according to example 4, see FIG. 7A. On top of that, hES-HEP cultured in maturation medium 11 supplemented with 1.5 M BIO (MMII+BIO)(example 9) performed even better than MMI+BIO cultures regarding Cyp 1A activity. This trend was demonstrated for all time points analysed, day 16-18, 20-21 and day 25. The Cyp1A activity increased during time in the two groups treated with BIO, suggesting maturation of hepatocyte-like cells over time in the presence of a GSK3 inhibitor. The Cyp 1A activity was supported by the finding of CYP1A2 and 1A1 gene expression levels similar to or greater than HepG2, see FIG. 7B. In addition, the protein CYP1A2 was detected in hESC-HEP cultures matured in the presence of BIO, see FIG. 7C. To summarise GSK3 inhibitors stimulate CYP1A-family members to be functionally expressed at both mRNA and protein levels in hESC-HEP. As CYP1A1 is expressed in the neonatal liver and CYP1A2 in the newborn and adult liver, the results point at GSK3 inhibitors to take an important part in differentiation and maturation of hESC into hepatocyte-like cells.

Example 16

hPS Cells from Feeder Free Cultures

(163) hESCs cultured under feeder free conditions were incubated in media supplemented with Activin A. The cells were then induced into hepatocytes by differentiating them to hepatic progenitor cells and then to more mature hepatocyte like cells. Cells cultured with and without the GSK3 inhibitor were compared for their hepatic profile and homogeneity. For culture details see FIG. 3 and examples 12, 13 and 14.

(164) From this study we could conclude that a GSK3 inhibitor (BIO) was significantly important for the differentiation, maturation, and homogeneity of the cultures. (See results in FIGS. 4A & B), since both metabolic activity (FIG. 4A) and hepatic gene marker expression (FIG. 4B) was higher in cells exposed to GSK-3 inhibitors and thus comparable with hESC-HEPs derived from hPS initially maintained on feeder cells. Confirmation that BIO treatment is affecting the Wnt pathway shown in FIG. 4C where beta-catenin is seen to translocate from cell membrane to nucleus upon with BIO, consistent with its signalling role in the Wnt pathway.

Example 17

Derivation of Hepatocyte Like Cells from hiPS Cells

(165) The culturing and derivation was performed as described in example 12-14 and outlined in FIG. 3, but with hiPS cells replacing the feeder free hPS cells.

(166) The undifferentiated hiPS cells were cultured in Activin A supplemented media to stimulate initial differentiation into partly differentiated cells. The hiPS derived partly differentiated cells were then passaged (to plates coated with 0.1% Gelatin or Matrigel 0.016 mM) and induced to hepatic progenitor cells and then to hepatocyte like cells in media with and without BIO supplementation, see FIG. 3 A)-B).

(167) The conclusion from this study was that the hiPS cells cultured in media supplemented with BIO responded significantly and became more mature compared to the cells that were grown in the absence of the GSK-3 inhibitor. This was concluded by analysing the expression profile of the cells by Q-PCR, immunocytochemistry and Activity Assay FIGS. 5A and B. These results verify and are unanimous with what we have observed for both hES cultured on mEF and hES cultured in a feeder free way (FIGS. 4A and B)

(168) The hiPS cells were cultured on mEFs until they were confluent. The cells were washed twice in PBS+/+ and treated with Activin A containing medium (See FIG. 3 a-b). The cells adapted an endoderm like morphology around day 3. When the majority of the cells were partly differentiated, the cells were exposed to media supplemented with factors inducing the cells into hepatocyte-like cells (See FIG. 3A)-B)) plus or minus BIO. Different matrix: Gelatin and Matrigel did not significantly affect the outcome. BIO affected the cells significantly in that they upregulated several markers for mature hepatocytes including CYPs (CYP1A2) (see FIGS. 5A and B). hESC-HEPs treated with BIO also showed greater metabolic activity (FIG. 5A).

(169) Results

(170) Conclusions of BIO in the Maturation Phase of the Hepatocyte-Like Cell Protocol:

(171) Increased CYP1A activity compared to controls without BIO. Increased mRNA of CYP1A1, CYP1A2, CYP3A4, CYP2C9, CYP7A1, MRP2, CD44, AFP, CK18, CK19 activity compared to controls without BIO.

(172) Conclusions of the use of a split medium (SM) and BIO in maturation phase of the hepatocyte-like cell protocol as of FIG. 3c). Illustrations of the results are shown in FIGS. 5A and B Increased CYP1A and CYP3A activity compared to controls without BIO. Increased mRNA of CD44, AFP, CK18, CK19, CYP1A1, CYP1A2, CYP3A4, CYP2C9, CYP7A1, Albumin, OATP2, A1AT, MRP2 activity compared to controls without BIO.

Example 18

Selective Differentiation Using a GSK3 Inhibitor (BIO) Results in a Homogenous Population of Hepatocytes Derived from hES Cells

(173) MEF cultured hESC differentiated into hepatocyte-like cells according to example 6 and 11 as outlined in FIG. 2 ii and iv (presence of BIO from day 3) was compared to hepatocyte-like cells differentiated by example 4 and 8D as outlined in FIG. 2 i and iii (absence of BIO and with BIO added from day 10-30, respectively) demonstrated purification of hepatocyte-like cells in the cultures treated with BIO from day 3 (example 6 and 11). Morphological observations are illustrated in FIGS. 8A-E. Cultures with no or low concentrations (1 M) of BIO resulted in highly heterogeneous cultures where hardly any hepatocyte-like cells were observed as shown in FIGS. 8A and B. Addition of 5 M of BIO day 3 resulted in a dramatic cell death to begin with. However, the surviving cells, DE-cells and/or anterior endodermal cells, day 4 were passaged into new wells and a highly purified and homogenous culture of hESC-HEP was appearing as the differentiation process proceeded, see FIG. 8C. An intermediate concentration of BIO (3.5 M) was tested in cultures from day 3 (Example 11) resulting in more purified hESC-HEP compared to untreated cultures (Example 9), FIGS. 8D and E respectively. In addition, a less dramatic cell death was observed compare to cultures grown in 5 M BIO from day 3. Thus it appears advantageous to add GSK-3 inhibitors at a later stage of differentiation to avoid massive cell death seen when it is added at day 3. Moreover the resulting purity of the cell populations (FIGS. 8A-E, purity table) also suggest that, at least for BIO, addition at a later stage (day 10+) gives greater final purity. Data suggest a role for GSK3 inhibitors in selection of DE-cells and/or anterior endodermal cells, a prerequisite for further differentiation into hepatoblasts and subsequently hepatocyte-like cells. In addition, GSK3 inhibitors at this stage may contribute/stimulate to hepatic induction of the competent endoderm.

Example 19

HDAC Inhibitors Potentiates Wnt-Signalling to Induce and Stimulate Hepatocyte Differentiation

(174) FIG. 9 shows data of functional CYP1A activity in hESC-HEP cultures (hepatocyte-like cells) differentiated in a maturation media, such as MMI, supplemented with an HDAC inhibitor, such as Sodium Butyrate (NaB, 1 mM) together with a GSK3 inhibitor, such as BIO (1.4 M) at day 21-25, following the protocol as described in example 14. Cell line SA002 from mEF cultures was used as starting material.

(175) Those hESC-HEP cultures were compared to parallel hESC-HEP cultures without NaB in the maturation media resulting in increased CYP1A activity in NaB containing cultures. Data suggests a role for HDAC inhibitors, e.g. NaB, to potentiate Wnt-signalling mediated transcription and effect on hepatocyte differentiation.

Example 20

Exposure of GSK3 Inhibitor at Early Hepatic Differentiation Improves Hepatic Gene Expression Profile of hESC-HEP

(176) hESC-HEP were derived from cell-line SA002 cultures on mEF-layer according to Example 9, FIG. 2, protocol iii (absence of BIO between day 3-10) or Example 11, FIG. 2, protocol iv (presence of 3.5 M BIO between day 3-10). Total RNA was collected and isolated from the two hES-HEP cultures at day 21, 24, 26 and 28 by using RNA isolation kit from Qiagene. Quantitative reverse transcriptase PCR, QrtPCR, by using Taqman probes, was performed for the following hepatic marker genes: phase I drug metabolizing enzymes; CYP (cytochrome P450) 3A4, 3A5, 3A7, 2C9, phase II drug metabolizing enzymes GSTA1 (glutation-S-transferas A1), UGT2B7 (UDP glucuronosyltransferase 2B7), phase III, transporters; MRP2 (multi-drug residence protein 2), BSEP (bile salt export pump), and general hepatic markers; A1AT (alpha-1 antitrypsin), ALB (albumin) and TAT (tyrosine-amino transferas). All data was normalised to the house-keeping gene CREB. RNA from HepG2 cultures was included and data is presented as fold change of HepG2. BSEP is an exception as HepG2 cells do not express the gene. Thus, BSEP expression is instead presented as fold change of a so called calibrator which contains RNA from different sources.

(177) Data is presented in FIGS. 10A to D and shows that all genes except for CYP3A7 are expressed at higher levels in hESC-HEP exposed to BIO from day 3 than in cultures where BIO was excluded from day 3-10. For CYP3A7 the opposite was observed. As CYP3A7 is a drug metabolizing enzyme which is mainly expressed prenatal and CYP3A4 in the newborn and adult liver, (Cyp 3A5 both pre and post natal and adult) the expression pattern of the CYP3A family members suggests a role for BIO early in the differentiation protocol to improve hepatic differentiation and contribute to a more maturation hESC-HEP culture by stimulating Wnt signalling. The improved expression levels of the hepatic markers in BIO exposed cultures are supporting the finding that GSK-3 inhibitors are important in early hepatic differentiation of hESC.

Example 21

Improvement of UGT Metabolic Activity in hESC-HEPs Exposed to GSK-3 Inhibition During Early Hepatic Differentiation

(178) Metabolic activity of hESC-HEP derived from hESCs cultured in feeder-free conditions (see Examples 12-14) was measured to determine the effect of GSK-3 inhibition during development and maturation by examining the activity of several UDP-glucuronyltransferases (UGTs) (enzymes that participate in the metabolism of many drugs). Activity of these UGTs was tested via several substrates, namely: -estradiol (UGT1A1, 3-glucuronide), 1-naphthol (UGT1A6), propofol (UGT1A9), and naloxone (UGT2B7). A non-specific control (Methylumbelliferone) was also included (see FIGS. 12A-B). hESC-HEPs were differentiated essentially according to Example 12, up to day 7. At this point, cells were treated to one of three protocols, either Protocol 1 (day 7-14 VH1 medium, day 14-25 Maturation medium BM2, BIO present only at days 14-25), Protocol 2 (day 7-25 Medium BM2, BIO present only at days 14-25) or Protocol 3 (day 7-14 Medium MMI, day 14-25 Maturation Medium MMII, BIO present days 14-25) with either +/GSK-3 inhibitor (in this case BIO) at a concentration of 1.4 M. A clear trend can be seen here, with most of the UGTs having greater activity in cells treated with GSK-3 inhibitor during development, and this trend is seen across all three differentiation protocols. For some UGTs (such as UGT1A1) the increase is much greater than for others, but the overall trend is that exposure of cells to GSK-3 inhibition leads to a more mature, metabolically active phenotype for all of the preferred differentiation methods.

Example 22

Induction of Early and Late Stage Hepatic Markers in hESC-HEPs by Modulation of Wnt-Signalling Using Non-810 GSK-3 Inhibitors at Later Stage Differentiation

(179) hESC-HEPs were derived essentially as described in Example 9, FIG. 2iii, but with alternative (non_BIO) GSK-3 inhibitors used to show that other GSK-3 inhibitors can be used interchangeably with BIO and can modulate the Wnt-signalling pathway during endoderm to hepatocyte differentiation to induce expression of hepatic marker genes. Furthermore, differentiating cells were exposed to GSK-3 inhibitors only during later (from day 10) stages of differentiation. Three alternative GSK-3 inhibitors were tested alongside BIO to determine effectiveness in inducing hepatic marker expression (SB216763, Kenpaullone and Indirubin-3-O) and results were compiled both for Phase I and Phase II enzymes and hepatic markers, with two negative controls (hESC-HEP not exposed to GSK-3 inhibition during differentiation, and undifferentiated hESC cells). Results are shown in FIG. 12A (Phase I enzyme markers) and FIG. 12B (Phase II enzymes and hepatic markers), and it can be seen that for Phase I enzymes, all GSK-3 substitutes in general seem to induce gene expression above the levels of the negative controls. Indeed, for most of the Phase I enzymes and hepatic markers, Kenpaullone seems to induce higher levels of expression than BIO. Results for Phase II enzymes and hepatic markers are somewhat more variable when different markers and different GSK-3 inhibitors are compared, but again in general it appears that the three alternative compounds tested can be used as feasible substitutes for BIO in modulating the Wnt-signalling pathway and producing mature hepatocyte-like cells. Expression of the housekeeping gene Creb shows little variation across the various samples and protocols. Equivalent results where hiPS cells were used instead of hESC-HEPS are shown in FIGS. 14A-C, and again support the finding that BIO can be substituted by other GSK-3 inhibitors.

Example 23

Induction of Early and Late Stage Hepatic Markers in hESC-HEPs by Modulation of Wnt-Signalling Using Non-810 GSK-3 Inhibitors at Both Mid and Later Stage Differentiation

(180) hESC-HEPs were derived essentially as described in Example 11 and FIG. 2iv, but with alternative (non_BIO) GSK-3 inhibitors used to show that other GSK-3 inhibitors can be used interchangeably with BIO and can modulate the Wnt-signalling pathway during endoderm to hepatocyte differentiation to induce expression of hepatic marker genes. Differentiating cells were exposed to GSK-3 inhibitors during both mid (days 3-9) and late (from day 10-23) stages of differentiation. Three alternative GSK-3 inhibitors were tested alongside BIO to determine effectiveness in inducing hepatic marker expression (SB216763, Kenpaullone and Indirubin-3-O) and results were compiled both for Phase I enzymes and Phase II enzymes and hepatic markers, with two negative controls (hESC-HEP not exposed to GSK-3 inhibition during differentiation, and undifferentiated hESC cells). During days 3-9 of differentiation, concentrations of GSK-3 inhibitors used were as follows: BIO 3.5 M, SB216763 34 nM, Kenpaullone 0.23 M and Indirubin-3-O 22 nM. For later stage differentiation (days 10-23) concentrations were changed to: BIO 1.5 M, SB216763 2.5 M, Kenpaullone 2.5 M and Indirubin-3-O 2.5 M Results are shown in FIG. 13A (Phase I enzymes) and FIG. 13B (Phase II enzymes), and it can be seen that for Phase I enzymes, GSK-3 substitutes can in general seem to induce hepatic marker gene expression above the levels of the negative controls and often above the level of that seen in BIO treatment. Indeed, for most of the Phase I enzymes, Kenpaullone seems to induce higher levels of expression than BIO. Results for Phase II enzymes and hepatic markers are somewhat more variable when different markers and different GSK-3 inhibitors are compared, but again in general it appears that the three alternative compounds tested can be used as feasible substitutes for BIO in modulating the Wnt-signalling pathway and producing mature hepatocyte-like cells. It is also apparent that modulation of Wnt-signalling at mid and late stages by GSK-3 inhibition is comparable to modulation during only late stage. Depending upon the result desired (expression of certain markers over others) then one method may be preferable over the other. Expression levels of the housekeeping gene Creb shows little variation across the various samples and protocols here. Equivalent results where hiPS cells were used instead of hESC-HEPS are shown in FIGS. 15A-C, and again support the finding that BIO can be substituted by other GSK-3 inhibitors.