ヒトiPS細胞由来肝幹前駆細胞の 大量増幅 ~大量の 薬研究におけるヒトiPS細胞由来分化誘導肝細胞の期待 ヒトiPS細胞から肝細胞への効率の良い分化誘導技術の開発、

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Text of ヒトiPS細胞由来肝幹前駆細胞の 大量増幅 ~大量の...

  • iPS

  • 4 OCT3/4, SOX2, KLF4, c-MYC

    iPS

    or

    iPS

    iPS

    iPS

  • iPS

    iPS

    95%

    iPS

  • iPS

    iPS

    iPS

    BMP4 FGF4

    3 days

    HGF OSM Dex

    11 days

    Activin A

    5-6 days

    iPS

    iPS

    iPS3

  • iPS/iPS

    1030 7090

    iPS

    iPS

  • iPS

    1030%

    iPS

    definitive endoderm differentiation

    hepatic specification

    hepatic expansion / maturation

    35-6

  • iPS

    Ad

    FOXA2HNF

    iPS

    BMP4 FGF4

    HGFOSMDex

    9-11 days

    Activin AbFGF

    6 days 3 days

    FOXA2+HNF1

    FOXA2+HNF1FOXA2

    CYP2D6 CYP3A4

    Rel

    ativ

    e G

    ene

    Exp

    ress

    ion

    0.0001 0.001 0.01 0.1 1 10

    iPS

    0.00001 0.0001 0.001 0.01 0.1 1 10

    iPS

    0 20 40 60 80

    100 120

    0 20 40 60 80 100

    Troglitazone

    cell

    viab

    ility

    (%)

    (M)

    iPS

    PS

    H24

    iPS

    iPS

  • Day 0

    Day 6

    Day 9

    iPS

    FGF4 + BMP4

    Activin A

    On laminin

    On laminin

    Day 1820

    On laminin

    HGF + OsM + DEX

    FOXA2

    FOXA2 + HNF1

    one step

  • ES/iPShepatoblast-like cell

    2 days

    hepatoblast-like

    cells 3 days 1 day

    stage II

    Activin A (100 ng/ml) BMP4 (30 ng/ml) FGF4 (20 ng/ml)

    3 days

    stage I

    ES /iPS

    Ad-FOXA2 (3,000 VP/cell)

    Eva Schmelzer et al. JEM. 2007

    200 m

    200 m

    AFP / DAPI

    human hepatic stem cells

    ES hepatoblast-like cells

    ES/iPS FOXA2 AFPhepatoblast-like cells

    80%

    # ES(H9)

    ES/iPSAFP Hepatoblast-like cells (HBC)

  • hepatoblast-like cells (HBC) non-hepatoblast-like cells (NHBC)

    AFP CD133 EpCAM CK8 CK18

    before HBCs NHBCs

    rela

    tive

    gene

    exp

    ress

    ion

    0

    1

    2 ** ** ** ** ** ** * ** * **

    HBCNHBC

    -14 -12 -10 -8 -6 -4 -2 0 2

    1 2 3 4 5 6 9 v 1 3 4 (C

    t val

    ue o

    f GAP

    DH

    )

    (Ct v

    alue

    of i

    nteg

    rin)

    integrin

    **

    ** before HBCs NHBCs

    HBCNHBC

    HBC

    HBCNHBC integrin 6

    integrin 6 HBCNHBC

    # before = ES9 # ESH9 # before1.0

    ES/iPSHBCs

  • before NHBCs% o

    f int

    egrin

    6-

    and

    1-p

    ositi

    ve c

    ells

    0

    25

    50

    75

    100

    HBCs

    hepatoblast-like cell(HBC)

    # ESH9

    ES/iPSHBCs61

  • LN111 LN211 LN411 LN511

    rela

    tive

    cell

    num

    ber before HBCs NHBCs

    0

    0.4

    0.8

    1.2 * **

    **

    LN111 LN211 LN411 LN511

    rela

    tive

    cell

    num

    ber

    control IgG anti-integrin 6 anti-integrin 1

    0

    0.4

    0.8

    1.2 *** ***

    * *** ***

    * *** *

    HBCNHBC

    HBCLN111 Integrin 61

    Integrin 6LN111 ES/iPSHBC

    HBCLN111 NHBCLN111 HBCLN111integrin 6

    Integrin 1 # before =

    ES9 # ESH9 # 1.0 # biolamina

    ES/iPSHBCs111 integrin 6integrin 1

  • 0

    2

    4

    6

    8 LN111 LN211 LN411 LN511

    rela

    tive

    cell

    num

    ber

    % o

    f AFP

    -pos

    itive

    cel

    ls

    day 16 day 9

    0

    20

    40

    60

    80

    100

    ES/iPSHBCs LN111

    AFP (FACS)

    ESHBCsLN111 LN111

    AFP # HBC P0 # day 101.0 # before passage = 9

    # HBC P0 = LN111

    ES/iPS511Rodin et al. Nat Biotech. 2010

    ES/iPS

    Dlk1+Tanimizu et al. JCS. 2004

    ES/iPSHBC111

  • ES/iPSHBCLN111

    matrigel-coated dish

    LN111-coated dish

    LN111-coated dish

    passage

    replace with fresh medium after 15 min

    from passage

    HBC NHBC

    dissociation by using accutase

    passage number

    rela

    tive

    cell

    num

    ber

    100 102 104 106 108 1010 1012

    0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

    human ES cells (H9) human iPS cells (Dotcom)

    ES/iPSHBCs LN111

    ES/iPSHBCsLN111 15

    AFP (FACS)

    LN111AFP

    passage number passage number

    % o

    f AFP

    -pos

    itive

    cel

    ls

    % o

    f AFP

    -pos

    itive

    cel

    ls

    human ES cells (H9)

    human iPS cells (Dotcom)

    0

    25

    50

    75

    100

    day 9 P1 P10 P15 0

    25

    50

    75

    100

    day 9 P1 P10 P15

    DMEM/F12 FBS (10%) HGF EGF Ins/TF/selenium Dex Nicotinamide

  • HBC hepatocyte -like cells

    HGF, OsM (20 ng/ml), DEX (10-6 M) matrigel

    14 days hepatocyte differentiation

    HBC P0

    HBC P10

    HBC clone %

    of A

    SGR

    1-po

    sitiv

    e ce

    lls

    0

    25

    50

    75

    100 ASGR1

    HBC

    # HBC P0 = LN111hepatoblast-like cells # HBC P10 = LN11110passagehepatoblast-like cells # HBC clone = hepatoblast-like cells

    CYP3A4 / DAPI

    HBC data not shown)

    iPS

    iPSiPS

    one step

    2013-80557

    CYP3A4

    rela

    tive

    gene

    exp

    ress

    ion

    0

    0.5

    1.0

    1.5

    HBC P10

    HBC clone

    HBC P0

    PH 48hr

    Takayama et al., Stem Cell Rep. (2013)

  • hepatic stem cell

    iPS

    iPS

  • iPS

  • iPS

    iPS

    JST

  • iPS

    iPS

  • 2013-80557

  • 2000- 2001- 2010- iPS 2010-

  • 565-0871 2-8

    TEL: 06-6879-4859 FAX: 06-6879-4205

    E-mail: [email protected]

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