US20140234963A1 - Efficient induction of definitive endoderm from pluripotent stem cells - Google Patents

Efficient induction of definitive endoderm from pluripotent stem cells Download PDF

Info

Publication number
US20140234963A1
US20140234963A1 US14/127,296 US201214127296A US2014234963A1 US 20140234963 A1 US20140234963 A1 US 20140234963A1 US 201214127296 A US201214127296 A US 201214127296A US 2014234963 A1 US2014234963 A1 US 2014234963A1
Authority
US
United States
Prior art keywords
cells
chir
concentration
stem cells
activin
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US14/127,296
Other languages
English (en)
Inventor
Nina Funa
Katja Hess
Jenny Ekberg
Henrik Semb
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novo Nordisk AS
Takara Bio Europe AB
Original Assignee
Novo Nordisk AS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novo Nordisk AS filed Critical Novo Nordisk AS
Priority to US14/127,296 priority Critical patent/US20140234963A1/en
Assigned to NOVO NORDISK A/S, CELLARTIS AB reassignment NOVO NORDISK A/S ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: SEMB, HENRIK, FUNA, Nina, HESS, Katja, EKBERG, JENNY
Publication of US20140234963A1 publication Critical patent/US20140234963A1/en
Assigned to TAKARA BIO EUROPE AB reassignment TAKARA BIO EUROPE AB CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: CELLARTIS AB
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0676—Pancreatic cells
    • C12N5/0678—Stem cells; Progenitor cells; Precursor cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0676—Pancreatic cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/16—Activin; Inhibin; Mullerian inhibiting substance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells

Definitions

  • the present invention relates to a method to differentiate pluripotent stem cells to a primitive streak cell population, in a stepwise manner for further maturation to definitive endoderm.
  • DE cells from pluripotent embryonic stem (ES) cells has been reported for both mouse and human in, e.g., WO2005/116073, WO2005/063971, and US 2006/0148081.
  • the generation of pancreatic endoderm (PE) cells from DE cells is necessary for the generation of insulin-producing beta cells for the treatment of diabetes.
  • CHIR is a glycogen synthase kinase 3 beta (Gsk3b) inhibitor and a known component of a defined tissue culture medium to maintain mouse embryonic stem cells in the pluripotent state (Ying et al Nature 453, 519-523).
  • Gsk3b has multiple targets but is mainly known to regulate degradation and/or nuclear transfer of beta-catenin.
  • the role of stabilized beta-catenin in PS formation from human embryonic stem cells (hESC) has been described using other glycogen synthase kinase 3 beta (Gsk3b) inhibitors such as BIO and Wnt3a.
  • CHIR is the most selective Gsk3b-inhibitor reported to date.
  • the present invention relates to the utilization of CHIR without AA, at a defined concentration range to treat pluripotent stem cells prior to AA-mediated definitive endoderm induction compared to the conventional induction protocol for obtaining DE (D'Amour protocol, as described in Kroon et al. 2008).
  • This sequential exposure, first to CHIR and then to AA, is essential and reflects successive PS formation (induced by CHIR) followed by more efficient and rapid DE formation by AA.
  • the present invention relates to the discrete and successive control of induction of first PS then followed by DE leading to an overall more efficient, with earlier peak of SOX17 expression and robust DE protocol.
  • Ctrl No Chir treatment D1. Cells are left in RPMI during priming period, followed by AA D2 and AA+serum replacement (B27) from D3 onwards.
  • FIG. 1 shows the CHIR DE protocol nomenclature and overview. The protocol has been confirmed in 7 different pluripotent stem cell lines: SA121, SA181, SA461, SA167 (human ESCs) and chIPS2, chIPS3, chIPS4 (human iPSCs).
  • FIG. 2 shows transcriptional expression of PS markers Brachyury (T), MIXL1, EOMES and Goosecoid (GSC) after 24 h of CHIR treatment (D1) in hES SA121 ( FIG. 2A ) and chIPS4 ( FIG. 2B ), compared to non-treated cells. T protein levels were also confirmed with ICC ( FIG. 2C ). T fold inductions after 24 h CHIR treatment have spanned between 500-100000 compared to undifferentiated cells (n>60). Bars show Log 10 values relative to undifferentiated cells D0.
  • FIG. 3 shows gene expression of DE markers (SOX17, CXCR4, FOXA2, CER) one day after replacing CHIR with ActivinA (AA) compared to with cells that were not pretreated with CHIR. Expression is shown both in hESC SA121 ( FIG. 3A ) and iPSC chIPS4 ( FIG. 3B ). Bars show Log 10 values relative to undifferentiated cells D0.
  • FIG. 4 shows hESC SA121 ( 4 A-B) and iPSC chIPS4 ( 4 C) cells treated with either CHIR (3 ⁇ M), BIO (0.5 uM), Wnt3a (200 ng/ml) or AA alone (100 ng/ml) for 24 h (D1) prior to 2 d AA treatment (D3). Additionally, D'Amour (protocol as described in Kroon et al 2008) was analyzed. Expression of Brachyury and SOX17 was analyzed. hES cells treated with AA or BIO for the first 24 h did not survive until day 3.
  • FIG. 5 shows gene expression levels of DE markers SOX17, FOXA2 and CXCR4 ( FIG. 5A-C ) and OCT4 ( FIG. 5E ) after CHIR induction compared to D'Amour and quantified ICC protein levels of SOX17/OCT4 in chIPS4 cells ( FIG. 5D , F). Corresponding % of cells positively stained for OCT4 and SOX17 (D1-3) are also indicated in Table 2.
  • chIPS/SA121 DE cells using either of the two protocols, were further differentiated towards pancreatic endoderm (PE) using a previously published PE differentiation protocol (Amen et al 2010). Cells were analyzed after seven days of PE induction using ICC for PDX1 protein levels.
  • PE pancreatic endoderm
  • FIG. 6 shows gene expression of SOX17 D3 in hES SA121 ( FIG. 6A ) or chIPS4 ( FIG. 6C ) treated with either only RPMI, AA and Wnt3a or CHIR D1 followed by treatment with either AA or a combination of AA and Wnt3a D2 and AA D3 ( FIG. 6A ).
  • Cells were also treated with either CHIR D1 and AA D2 (“CHIR”), D'Amour, or CHIR on top of D'Amour and were analyzed for SOX17 expression ( FIG. 6B ) and morphology ( FIG. 6C ).
  • CHIR D1 and AA D2 (“CHIR”)
  • D'Amour D'Amour
  • CHIR CHIR on top of D'Amour
  • FIG. 7 shows that CHIR was titrated at concentrations between 1-7 uM and analysed after 24 h (D1) and after 2 d of following AA treatment (D3). CHIR 0.5-1 uM and CHIR 7 uM did not survive D3. Brachyury expression was analysed after 24 h treatment in SA121 ( FIG. 7A ) and chIPS cells ( FIG. 7C ). SOX17 expression was analysed D3 in SA121 ( FIG. 7B ) and chIPS cells ( FIG. 7D )).
  • FIG. 8 shows gene expression of SOX17 D3 after CHIR pre-treatment or after the D'Amour protocol in the mTeSR system ( FIG. 8 ).
  • FIG. 9 shows gene expression levels of markers Brachyury and SOX17 at D0, D2 and D3 when cultured on MEFfeeders.
  • the present invention also relates to a method for differentiation of stem cells into definitive endoderm comprising the steps of:
  • the present invention relates to a faster and more pronounced peak of SOX17 expression and a more robust method of differentiating human pluripotent stem cells to obtain definitive endoderm.
  • the present invention relates to a method for differentiation of stem cells into definitive endoderm comprising the steps of incubating pluripotent stem cells in a medium comprising at least 2 ⁇ M CHIR and subsequently incubating these cells in a medium comprising Activin A (AA).
  • the present inventors have found that using CHIR alone for 24 h prior to further addition of AA, induced a significant and dose dependent upregulation of PS markers such as Brachyury, Mixl1, Eomes and Goosecoid (GSC) during these 24 h.
  • PS markers such as Brachyury, Mixl1, Eomes and Goosecoid (GSC) during these 24 h.
  • the present inventors have surprisingly found that in the subsequent incubation with AA to induce DE, DE induction as measured by SOX17 expression, peaked at an earlier time point and with higher fold change when compared to cultures without CHIR pre-incubation or when compared to the conventional D'Amour protocol (Novocell, Nature Biotec 2006, 2008).
  • the present inventors surprisingly found that the method of the present invention was more efficient and showed a faster induction of Sox17 mRNA expression than other known protocols for DE Induction.
  • the CHIR-mediated effect has been reproduced both in other culture systems (mTeSR media and conventional ES media on mouse embryonic feeder (MEF) cells) and across different cell lines (SA121, SA181, SA461, SA167, chIPS2, chIPS3, chIPS4), showing that the effect is not dependent on the cell line or the DEF media culture system.
  • Human embryonic stem cells may be derived from single blastomeres without the destruction of the embryo (Klimanskaya et al. 2006; Chung et al. 2008; Geens et al. 2009).
  • Cell lines chIPS2, chIPS3, chIPS4 are iPS cell lines.
  • CHIR CHIR promotes high cell densities after AA is added. Dose dependency of CHIR in PS induction will allow future optimization of possible pre-patterning influences when initiating AA treatment. The subsequent order of added factors decreases the amount of signaling pathways that are activated in parallel. Minimizing the amount of factors that may influence the efficiency should promote robustness and reproducibility.
  • the present inventors have surprisingly found that the CHIR-priming step is unique for DE induction and was superior when compared to BIO or Wnt3a when it comes to efficiency peak of SOX17 expression and robustness.
  • hESCs Human embryonic stem cells
  • iPSCs human induced pluripotent stem cells
  • the present inventors have surprisingly found the CHIR-priming step resulted in a remarkably fast induction of SOX17 by AA peaking already after 1 day which is in contrast to AA alone where the peak is seen only after 3-4 days.
  • the sequential incubation with CHIR followed by AA is essential for DE formation to favourable levels.
  • the pancreatic endocrine cells obtainable by the method according to the invention are insulin producing cells, optionally together with cells differentiated towards glucagon, somatostatin, pancreatic polypeptide, and/or ghrelin producing cells.
  • insulin producing cells refers to cells that produce and store or secrete detectable amounts of insulin.
  • Insulin producing cells can be individual cells or collections of cells.
  • the cell population comprising pancreatic cells is obtained from a somatic cell population.
  • the somatic cell population has been induced to de-differentiate in to an embryonic-like stem (ES, e.g., a pluripotent) cell.
  • ES embryonic-like stem
  • IPS induced pluripotent stem cells
  • the cell population comprising pancreatic cells is obtained from embryonic stem (ES, e.g., pluripotent) cells.
  • ES embryonic stem
  • the cell population comprising pancreatic cells is pluripotent cells such as ES like-cells.
  • the cell population comprising pancreatic cells is embryonic differentiated stem (ES or pluripotent) cells. Differentiation takes place in embryoid bodies and/or in monolayer cell cultures or a combination thereof.
  • ES embryonic differentiated stem
  • the cell population is a population of stem cells.
  • the cell population is a population of stem cells differentiated to the pancreatic endocrine lineage.
  • Stem cells are undifferentiated cells defined by their ability at the single cell level to both self-renew and differentiate to produce progeny cells, including self-renewing progenitors, non-renewing progenitors, and terminally differentiated cells. Stem cells are also characterized by their ability to differentiate in vitro into functional cells of various cell lineages from multiple germ layers (endoderm, mesoderm and ectoderm), as well as to give rise to tissues of multiple germ layers following transplantation and to contribute substantially to most, if not all, tissues following injection into blastocysts.
  • Stem cells are classified by their developmental potential as: (1) totipotent, meaning able to give rise to all embryonic and extra embryonic cell types; (2) pluripotent, meaning able to give rise to all embryonic cell types; (3) multi-potent, meaning able to give rise to a subset of cell lineages, but all within a particular tissue, organ, or physiological system (for example, hematopoietic stem cells (HSC) can produce progeny that include HSC (self-renewal), blood cell restricted oligopotent progenitors and all cell types and elements (e.g., platelets) that are normal components of the blood); (4) oligopotent, meaning able to give rise to a more restricted subset of cell lineages than multi-potent stem cells; and (5) unipotent, meaning able to give rise to a single cell lineage (e.g., spermatogenic stem cells).
  • HSC hematopoietic stem cells
  • a protocol for obtaining pancreatic cells from stem cells is exemplified by, but not limited to, the protocols described in D'Amour, K. A. et al. (2006), Nat Biotechnol 24, 1392-401; Jiang, J. et al. (2007), Stem Cells 25, 1940-53; and Kroon, E. et al. (2008), Nat Biotechnol 26, 443-452.
  • a protocol for obtaining pancreatic cells from somatic cells or somatic cells induced to de-differentiate into pluripotent cells such as ES like-cells is exemplified by, but not limited to, the protocols described in Aoi, T. et al. (2008), Science 321 (no. 5889), 699-702; D'Amour, K. A. et al. (2006), Nat Biotechnol 24, 1392-401; Jiang, J. et al. (2007), Stem Cells 25, 1940-53; Kroon, E. et al. (2008), Nat Biotechnol 26, 443-452; Takahashi, K. et al. (2007), Cell 131, 861-72; Takahashi, K., and Yamanaka, S. (2006), Cell 126, 663-76; and Wernig, M. et al. (2007), Nature 448, 318-24.
  • differentiate refers to a process where cells progress from an undifferentiated state to a differentiated state, from an immature state to a less immature state or from an immature state to a mature state.
  • characteristics markers like Pdx1, Nkx6.1, and Ptf1a.
  • Mature or differentiated pancreatic cells do not proliferate and do secrete high levels of pancreatic endocrine hormones or digestive enzymes.
  • fully differentiated beta cells secrete insulin at high levels in response to glucose. Changes in cell interaction and maturation occur as cells lose markers of undifferentiated cells or gain markers of differentiated cells.
  • differentiation factor refers to a compound added to pancreatic cells to enhance their differentiation to mature endocrine cells also containing insulin producing beta cells.
  • exemplary differentiation factors include hepatocyte growth factor, keratinocyte growth factor, exendin-4, basic fibroblast growth factor, insulin-like growth factor-1, nerve growth factor, epidermal growth factor platelet-derived growth factor, and glucagon-like peptide 1.
  • differentiation of the cells comprises culturing the cells in a medium comprising one or more differentiation factors.
  • human pluripotent stem cells refers to cells that may be derived from any source and that are capable, under appropriate conditions, of producing human progeny of different cell types that are derivatives of all of the 3 germinal layers (endoderm, mesoderm, and ectoderm). hPS cells may have the ability to form a teratoma in 8-12 week old SCID mice and/or the ability to form identifiable cells of all three germ layers in tissue culture. Included in the definition of human pluripotent stem cells are embryonic cells of various types including human blastocyst derived stem (hBS) cells in 30 literature often denoted as human embryonic stem (hES) cells, (see, e.g., Thomson et al.
  • hBS human blastocyst derived stem
  • hES human embryonic stem
  • hPS cells suitable for use may be obtained from developing embryos.
  • suitable hPS cells may be obtained from established cell lines and/or human induced pluripotent stem (hiPS) cells.
  • hiPS cells refers to human induced pluripotent stem cells.
  • the term “blastocyst-derived stem cell” is denoted BS cell, and the human form is termed “hBS cells”.
  • BS cell the human form
  • the pluripotent stem cells used in the present invention can thus be embryonic stem cells prepared from blastocysts, as described in e.g. WO 03/055992 and WO 2007/042225, or be commercially available hBS cells or cell lines.
  • any human pluripotent stem cell can be used in the present invention, including differentiated adult cells which are reprogrammed to pluripotent cells by e.g. the treating adult cells with certain transcription factors, such as OCT4, SOX2, NANOG, and LIN28 as disclosed in Yu, et al., 2007, Takahashi et al. 2007 and Yu et al 2009.
  • feeder cells are intended to mean supporting cell types used alone or in combination.
  • the cell type may further be of human or other species origin.
  • the tissue from which the feeder cells may be derived include embryonic, fetal, neonatal, juvenile or adult tissue, and it further includes tissue derived from skin, including foreskin, umbilical chord, muscle, lung, epithelium, placenta, fallopian tube, glandula, stroma or breast.
  • the feeder cells may be derived from cell types pertaining to the group consisting of human fibroblasts, fibrocytes, myocytes, keratinocytes, endothelial cells and epithelial cells.
  • Examples of specific cell types that may be used for deriving feeder cells include embryonic fibroblasts, extra embryonic endodermal cells, extra embryonic mesoderm cells, fetal fibroblasts and/or fibrocytes, fetal muscle cells, fetal skin cells, fetal lung cells, fetal endothelial cells, fetal epithelial cells, umbilical chord mesenchymal cells, placental fibroblasts and/or fibrocytes, placental endothelial cells,
  • MEF cells is intended to mean mouse embryonic fibroblasts.
  • CHIR glycogen synthase kinase 3 beta
  • bFGF basic fibroblast growth factor (FGF2)
  • Gsk3b glycogen synthase kinase 3 beta
  • hESC human embryonic stem cells
  • hIPSC human induced pluripotent cells
  • hPSC human pluripotent stem cells
  • RNA ribonucleic acid
  • SOX17 SRY (sex determining region Y)-box 17
  • the DE protocol was confirmed in two different feeder free (DEF, mTeSR) culture systems and one feeder dependent (MEF-ES) culture system.
  • hES cells SA121 and chIPS4 were grown on human fibronectin (Sigma) in DEF culture media (Cellartis) with 30 ng/ml bFGF (Invitrogen) and 10 ng/ml Noggin (Peprotech) in 6-96 well plates.
  • Cells were single cell passaged with Rock inhibitor Y-27632 (Calbiochem) and seeded at a density of 40000 cells/cm2 for experiments. Experiments were initiated 4 d after passage.
  • hES cells were cluster passaged from feeders (MEFs) to Matrigel (BD Biosciences) in mTeSR1 media (Cell Signaling Technologies) and passaged further with Dispase (BD Biosciences) according to the culture system protocol. DE was initiated once clusters started to touch each other
  • hES cells (SA121) were passaged on gelatine-coated plates pre-seeded with MEFs in hES media (KO-DMEM, PEST, Glutamax, NEAA, 2-mercaptoethanol, KO serum replacement, 10 ng/ml bFGF). DE was initiated at 80% confluency.
  • FIG. 1 Overview of the CHIR Definitive Endoderm protocol can be found in FIG. 1 .
  • the protocol has been confirmed in 7 different cell lines: SA121, SA181, SA461, SA167 (hESC) and chIPS2, chIPS3, chIPS4 (hiPSC).
  • Day 3&4 mediums were premixed (100 ng/ml Activin A and 2% B27 in prewarmed RPMI+0.1% PEST) and gently added to cell culture.
  • RNA samples were collected after 24 h pre-treatment (D1), after 1 day of AA treatment (D2) and after 1-2 d of AA+B27 treatment (D3-4). Total RNA was extracted with the Rneasy Plus Mini kit (Qiagen) and quantitative real-time PCR was performed using the StepOnePLus system (Applied Biosystems).
  • Undifferentiated SA121 hES and chIPS4 iPS cells in DEF media were cultured either in RPMI without CHIR (Ctrl D1) or treated for 24 h with CHIR in RPMI (CHIR D1). The cells were then washed to ensure absence of CHIR and then both conditions were treated with 1 d AA (Ctrl and CHIR d2). After 1 d CHIR treatment, PS markers such as Brachyury (T), MIXL1, EOMES and GSC were highly upregulated compared to non pre-treated cells ( FIG. 2A-C ). T fold inductions after 24 h CHIR treatment spanned between 500-100000 compared to undifferentiated cells (d0). T protein levels were also confirmed with ICC ( FIG. 2C ).
  • SOX17 expression peaked already after one day of AA treatment when cells were subjected to CHIR for 24 h before AA induction.
  • Cells in AA-containing media that were not pre-treated with CHIR (ctrl) did not show same speed or efficiency in Sox17 induction.
  • CHIR has a direct capacity to induce PS markers after 24 h treatment (D1) and resulted in a remarkably higher ability for AA to induce DE when added (D2). Brachyury was greatly repressed once AA was added indicating that the cells were differentiating in a highly stepwise order.
  • OCT4 and SOX17 protein ICC quantifications in undifferentiated hES SA121/chIPS4 cells OCT4 SOX17 SA121 chIPS4 SA121 chIPS4 ICC % D'Amour CHIR D'Amour CHIR D'Amour CHIR D'Amour CHIR Undiff 97.5 98.3 0 0 D1 91.9 97.7 94.3 91.2 0 0 8.3 0.9 D2 55.2 2.4 14 3.1 61.6 72.1 75.2 68.8 D3 44.1 0.2 26.2 3.7 80.4 93.1 77.6 91.5
  • Table 2 shows D1-3 of DE induction according to D'Amour (protocol as described in Kroon et al 2008) and cells treated with 3 uM CHIR (CHIR D1) prior to addition of ActivinA (CHIR D2-3).
  • hES SA121 and ChIPS4 cells were treated with either CHIR (3 ⁇ M), BIO (0.5 uM), Wnt3a (200 ng/ml) or AA alone (100 ng/ml) for 24 h prior to AA addition to show CHIR specificity (d1). Additionally, D'Amour (protocol as described in Kroon et al 2008) was tested in parallel. CHIR treatment significantly upregulated Brachyury expression after 24 h (FIG. 4 A,C). After AA addition (D2), Brachyury was reduced and SOX17 was upregulated ( FIG. 4B ). hES cells treated with AA or BIO did not survive until day 3.
  • Undifferentiated ChIPS4 cells (d0) were either pre-treated for 24 h with CHIR (3 uM) or directly exposed to AA (100 ng/ml) and Wnta3a (25 ng/ml) according to Kroon et al 2008. The cells were then washed and AA was added for 1-4 d. See Table 1 for detailed setup.
  • CHIR treatment was superior in upregulating both T (mRNA D1) and SOX17 after CHIR pre-treatment (mRNA, ICC D3) compared to D'Amour ( FIG. 5 and Table 2).
  • mRNA D1 mRNA D1
  • SOX17 SOX17 after CHIR pre-treatment
  • ICC D3 D'Amour
  • the most striking difference between the protocols could be seen in terms of morphology with a much higher degree of cell death during D'Amour treatment.
  • chIPS4 cells treated with either the CHIR- or D'Amour-protocol were fixed and stained using a SOX17-specific ab and the images were quantified in respect of total amounts and the percentage of SOX17-positive cells. As shown in FIG.
  • chIPS4 cells differentiated to DE were further differentiated towards pancreatic endoderm (PE) by using a previously published PE differentiation protocol (Amen et al 2010). Seven days after the induction of PE differentiation the cells were fixed and analyzed by ICC using a PDX1 specific antibody. The results showed very modest PDX1-staining in the D'Amour-treated cells whereas the staining was evident in the CHIR pre-treated cells (data not shown). In parallel, mRNA was collected from the cells and the PDX1 expression was analyzed by real-time PCR.
  • Cells were pre-treated 1 d with either RPMI (ctrl), CHIR 3 uM, CHIR 3 uM+AA 100 ng/ml or 100 ng/ml AA+25 ng/ml Wnt3a (D'Amour) prior to addition of AA+/ ⁇ Wnt3a (25 ng/ml) d2 and AA d3 (Table 3).
  • B27 was added in Ctrl and CHIR d3 and FBS to D'Amour d2-3.
  • CHIR pretreatment replaces the requirement of both Wnt3a and AA during the first day of differentiation toward DE and also has a stabilizing effect leading to a more robust protocol.
  • CHIR and exogenous AA may have counteracting effects that reduce overall efficiency and/or target different cell populations differently, resulting in higher levels of heterogeneity within the cell culture and increased inter-experimental variability. This suggests that PS induction/transition before adding AA to the media is an important and novel step to enhance protocol efficiency and robustness.
  • Chir was titrated at concentrations between 1-7 uM and analyzed after 24 h Chir treatment (D1) and after 2 d of following AA treatment (D3). CHIR 0.5-1 uM and CHIR 7 uM did not survive d3.
  • the data indicates that PS formation is delayed at Chir concentrations under 1 uM, unabling further progression toward DE when AA is added.

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Developmental Biology & Embryology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Reproductive Health (AREA)
  • Gynecology & Obstetrics (AREA)
  • Transplantation (AREA)
  • Diabetes (AREA)
  • Public Health (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Hematology (AREA)
  • Endocrinology (AREA)
  • Emergency Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Veterinary Medicine (AREA)
  • Obesity (AREA)
  • General Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Materials For Medical Uses (AREA)
US14/127,296 2011-06-21 2012-06-21 Efficient induction of definitive endoderm from pluripotent stem cells Abandoned US20140234963A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US14/127,296 US20140234963A1 (en) 2011-06-21 2012-06-21 Efficient induction of definitive endoderm from pluripotent stem cells

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
EP11170713 2011-06-21
EP11170713.9 2011-06-21
US201161501351P 2011-06-27 2011-06-27
US14/127,296 US20140234963A1 (en) 2011-06-21 2012-06-21 Efficient induction of definitive endoderm from pluripotent stem cells
PCT/EP2012/062013 WO2012175633A1 (en) 2011-06-21 2012-06-21 Efficient induction of definitive endoderm from pluripotent stem cells

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2012/062013 A-371-Of-International WO2012175633A1 (en) 2011-06-21 2012-06-21 Efficient induction of definitive endoderm from pluripotent stem cells

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US15/705,705 Continuation US10487313B2 (en) 2011-06-21 2017-09-15 Efficient induction of definitive endoderm from pluripotent stem cells

Publications (1)

Publication Number Publication Date
US20140234963A1 true US20140234963A1 (en) 2014-08-21

Family

ID=47422055

Family Applications (3)

Application Number Title Priority Date Filing Date
US14/127,296 Abandoned US20140234963A1 (en) 2011-06-21 2012-06-21 Efficient induction of definitive endoderm from pluripotent stem cells
US15/705,705 Active US10487313B2 (en) 2011-06-21 2017-09-15 Efficient induction of definitive endoderm from pluripotent stem cells
US16/694,040 Abandoned US20200190477A1 (en) 2011-06-21 2019-11-25 Efficient induction of definitive endoderm from pluripotent stem cells

Family Applications After (2)

Application Number Title Priority Date Filing Date
US15/705,705 Active US10487313B2 (en) 2011-06-21 2017-09-15 Efficient induction of definitive endoderm from pluripotent stem cells
US16/694,040 Abandoned US20200190477A1 (en) 2011-06-21 2019-11-25 Efficient induction of definitive endoderm from pluripotent stem cells

Country Status (6)

Country Link
US (3) US20140234963A1 (enrdf_load_stackoverflow)
EP (1) EP2723852B1 (enrdf_load_stackoverflow)
JP (1) JP6312591B2 (enrdf_load_stackoverflow)
CN (2) CN103890167A (enrdf_load_stackoverflow)
ES (1) ES2902650T3 (enrdf_load_stackoverflow)
WO (1) WO2012175633A1 (enrdf_load_stackoverflow)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021099532A1 (en) 2019-11-22 2021-05-27 Novo Nordisk A/S Spin-aggregated neural microspheres and the application thereof

Families Citing this family (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103890167A (zh) 2011-06-21 2014-06-25 诺沃—诺迪斯克有限公司 自多潜能干细胞有效诱导定形内胚层
WO2014033322A1 (en) 2012-09-03 2014-03-06 Novo Nordisk A/S Generation of pancreatic endoderm from pluripotent stem cells using small molecules
GB201317869D0 (en) * 2013-10-09 2013-11-20 Cambridge Entpr Ltd In vitro production of foregut stem cells
US11274280B2 (en) 2016-02-24 2022-03-15 Novo Nordisk A/S Generation of functional beta cells from human pluripotent stem cell-derived endocrine progenitors
US20190071645A1 (en) * 2017-09-07 2019-03-07 Sergio Mora Efficient differentiation of human stem cells to definitive endoderm
BR112020004428A2 (pt) 2017-09-11 2020-09-08 Novo Nordisk A/S métodos de criopreservação de agregados de células endócrinas pancreáticas e de enriquecimento de agregados de células endócrinas, células endócrinas, composição contendo células endócrinas, medicamento, e, dispositivo.
AU2018439389A1 (en) 2018-08-30 2021-02-18 Novo Nordisk A/S Generation of functional beta cells from human pluripotent stem cell-derived endocrine progenitors
US20220177849A1 (en) 2019-04-08 2022-06-09 Novo Nordisk A/S Generation of pancreatic endoderm from stem cell derived definitive endoderm
US20230313145A1 (en) 2020-08-28 2023-10-05 Novo Nordisk A/S Method for screening in vitro population of stem cell derived beta like cells and novel markers thereof
WO2024008810A1 (en) 2022-07-06 2024-01-11 Novo Nordisk A/S Differentiation of stem cells to pancreatic endocrine cells
WO2024151541A1 (en) 2023-01-09 2024-07-18 Sana Biotechnology, Inc. Type-1 diabetes autoimmune mouse
WO2024243236A2 (en) 2023-05-22 2024-11-28 Sana Biotechnology, Inc. Methods of delivery of islet cells and related methods
WO2025093467A1 (en) 2023-10-31 2025-05-08 Novo Nordisk A/S Methods for preventing unwanted cells arising during differentiation of human pluripotent stem cells
EP4491712A1 (en) 2024-03-20 2025-01-15 Novo Nordisk A/S Method for detecting contaminant cells in a stem cell derived cell population

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5942435A (en) * 1993-05-14 1999-08-24 The Board Of Trustees Of The University Of Illinois Transgenic swine compositions and methods
WO2009027654A1 (en) * 2007-08-24 2009-03-05 University Court Of The University Of Edinburgh Regionalised endoderm cells and uses thereof

Family Cites Families (53)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999065897A1 (en) 1998-06-19 1999-12-23 Chiron Corporation Inhibitors of glycogen synthase kinase 3
WO2002092756A2 (en) 2001-05-15 2002-11-21 Rappaport Family Institute For Research In The Medical Sciences Insulin producing cells derived from human embryonic stem cells
KR101008868B1 (ko) 2001-12-07 2011-01-17 제론 코포레이션 인간 배아 줄기세포 유래의 섬세포
EP1461421A2 (en) 2001-12-28 2004-09-29 Cellartis AB A method for the establishment of a pluripotent human blastocyst-derived stem cell line
CA2549605C (en) 2003-12-23 2013-05-07 Cythera, Inc. Definitive endoderm
US7625753B2 (en) 2003-12-23 2009-12-01 Cythera, Inc. Expansion of definitive endoderm cells
CA2564114C (en) 2004-04-27 2018-10-09 Cythera, Inc. Pdx1 expressing endoderm
WO2007042225A2 (en) 2005-10-07 2007-04-19 Cellartis Ab A method for obtaining a xeno-free hbs cell line
EP1999253B1 (en) 2006-03-02 2019-05-22 Viacyte, Inc. Endocrine precursor cells, pancreatic hormone-expressing cells and methods of production
GB0615327D0 (en) 2006-03-30 2006-09-13 Univ Edinburgh Culture medium containing kinase inhibitors and uses thereof
EP2021462B1 (en) 2006-04-28 2019-01-09 Lifescan, Inc. Differentiation of human embryonic stem cells
US8741643B2 (en) * 2006-04-28 2014-06-03 Lifescan, Inc. Differentiation of pluripotent stem cells to definitive endoderm lineage
AU2007254766A1 (en) 2006-06-02 2007-12-13 University Of Georgia Research Foundation, Inc. Pancreatic and liver endoderm cells and tissue by differentiation of definitive endoderm cells obtained from human embryonic stems
US9175260B2 (en) * 2007-01-30 2015-11-03 TheUniversity of Georgia Research Foundation, Inc. Early mesoderm cells, a stable population of mesendoderm cells that has utility for generation of endoderm and mesoderm lineages and multipotent migratory cells (MMC)
WO2008133904A1 (en) 2007-04-23 2008-11-06 Stowers Institute For Medical Research Methods and compositions for stem cell self-renewal
US9080145B2 (en) * 2007-07-01 2015-07-14 Lifescan Corporation Single pluripotent stem cell culture
BRPI0814425A2 (pt) 2007-07-18 2014-10-21 Lifescan Inc Diferenciação de células-tronco embrionárias humanas
US8951792B2 (en) 2007-07-20 2015-02-10 Cellartis Ab Methods for making definitive endoderm hepatocyte (DE-hep) progenitor cells
EP2610336A1 (en) * 2007-07-31 2013-07-03 Lifescan, Inc. Differentiation of human embryonic stem cells
PL2185691T3 (pl) 2007-07-31 2018-08-31 Lifescan, Inc. Różnicowanie pluripotencjalnych komórek macierzystych poprzez zastosowanie ludzkich komórek odżywczych
US8623648B2 (en) 2008-04-24 2014-01-07 Janssen Biotech, Inc. Treatment of pluripotent cells
US7939322B2 (en) 2008-04-24 2011-05-10 Centocor Ortho Biotech Inc. Cells expressing pluripotency markers and expressing markers characteristic of the definitive endoderm
EP2297298A4 (en) 2008-05-09 2011-10-05 Vistagen Therapeutics Inc PANCREATIC ENDOCRINE PROGENITOR CELLS FROM PLURIPOTENT STEM CELLS
DK2297319T3 (en) * 2008-06-03 2015-10-19 Viacyte Inc GROWTH FACTORS FOR PREPARING DEFINITIVE ENDODERM
US20090298178A1 (en) * 2008-06-03 2009-12-03 D Amour Kevin Allen Growth factors for production of definitive endoderm
MX2011000123A (es) 2008-06-30 2011-02-25 Centocor Ortho Biotech Inc Diferenciacion de las celulas madre pluripotentes.
KR20180018839A (ko) 2008-06-30 2018-02-21 얀센 바이오테크 인코포레이티드 만능 줄기 세포의 분화
US20100028307A1 (en) 2008-07-31 2010-02-04 O'neil John J Pluripotent stem cell differentiation
US8278105B2 (en) * 2008-09-09 2012-10-02 University Of Southern California Induction, propagation and isolation of liver progenitor cells
CN101684454B (zh) * 2008-09-25 2012-08-15 中国科学院上海生命科学研究院 一种定型内胚层细胞的制备和分离方法
ES2634445T3 (es) 2008-10-31 2017-09-27 Janssen Biotech, Inc. Diferenciación de las células madre embrionarias humanas con el linaje endocrino pancreático
MX349178B (es) * 2008-10-31 2017-07-17 Centocor Ortho Biotech Inc Diferenciación de células madre embrionarias humanas al linaje endocrino pancreático.
JP2012508584A (ja) 2008-11-14 2012-04-12 ヴィアサイト，インコーポレイテッド ヒト多能性幹細胞由来膵臓細胞のカプセル化
WO2010091241A2 (en) 2009-02-06 2010-08-12 President And Fellows Of Harvard College Compositions and methods for promoting the generation of definitive endoderm
EP3904505A1 (en) 2009-04-22 2021-11-03 Viacyte, Inc. Cell compositions derived from dedifferentiated reprogrammed cells
JP2012527903A (ja) 2009-05-27 2012-11-12 ザ・ソーク・インスティチュート・フォー・バイオロジカル・スタディーズ 遺伝的に是正された無病の誘導多能性幹細胞の作製法
AU2010276438B2 (en) 2009-07-20 2015-06-11 Janssen Biotech Inc. Differentiation of human embryonic stem cells
CN102482640B (zh) 2009-07-20 2015-03-11 詹森生物科技公司 人胚胎干细胞的分化
CN102712902B (zh) 2009-12-23 2019-01-08 詹森生物科技公司 人胚胎干细胞的分化
EP2516625B1 (en) 2009-12-23 2024-06-26 Janssen Biotech, Inc. Differentiation of human embryonic stem cells
CA2785966C (en) 2009-12-29 2020-10-27 Takeda Pharmaceutical Company Limited Method for manufacturing pancreatic-hormone-producing cells
CA2791476C (en) 2010-03-01 2020-06-30 Janssen Biotech, Inc. Methods for purifying cells derived from pluripotent stem cells
US9719068B2 (en) 2010-05-06 2017-08-01 Children's Hospital Medical Center Methods and systems for converting precursor cells into intestinal tissues through directed differentiation
KR101986176B1 (ko) 2010-05-12 2019-06-05 얀센 바이오테크 인코포레이티드 인간 배아 줄기 세포의 분화
WO2011160066A1 (en) 2010-06-17 2011-12-22 Regents Of The University Of Minnesota Production of insulin producing cells
US20120039955A1 (en) 2010-08-12 2012-02-16 Janssen Biotech, Inc. Treatment of Diabetes with Pancreatic Endocrine Precursor Cells
WO2012030539A2 (en) 2010-08-31 2012-03-08 Janssen Biotech, Inc. Differentiation of human embryonic stem cells
PH12013500367A1 (en) 2010-08-31 2013-04-01 Janssen Biotech Inc Differentiation of human embryonic stem cells
US9062292B2 (en) 2010-09-13 2015-06-23 Enzo Life Sciences Inc. Mutant T7 polymerases
WO2012070014A2 (en) 2010-11-26 2012-05-31 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Identification of novel cell surface markers for pancreatic progenitor cells and definite endodermal cells
EP2649177B1 (en) 2010-12-08 2018-09-05 ViaCyte, Inc. Agents and methods for inhibiting human pluripotent stem cell growth
WO2012081029A1 (en) 2010-12-15 2012-06-21 Kadimastem Ltd. Insulin producing cells derived from pluripotent stem cells
CN103890167A (zh) 2011-06-21 2014-06-25 诺沃—诺迪斯克有限公司 自多潜能干细胞有效诱导定形内胚层

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5942435A (en) * 1993-05-14 1999-08-24 The Board Of Trustees Of The University Of Illinois Transgenic swine compositions and methods
WO2009027654A1 (en) * 2007-08-24 2009-03-05 University Court Of The University Of Edinburgh Regionalised endoderm cells and uses thereof

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
Bone et al., (e-Published 5/24/2011, J. Cell Sci., Vol. 124, pgs. 1992-2000) *
Bone et al., e-Published 5/24/2011, J. Cell Sci., Vol. 124, pgs. 1992-2000. *
Brevini et al., 2010, Theriogenology, Vol. 74, pgs. 544-550 *
Cohen et al. (2004, Nature Review, Drug Discovery, Vol. 3, pgs. 479-487). *
Cohen et al., 2004, Nature Review, Drug Discovery, Vol. 3, pgs. 479-487. *
D’Amour et al. (2006, Nature Biotechnology, Vol. 24(11) pgs. 1392-1401) *
D'Amour et al., (2006, Nature Biotech., Vol. 24(11), pgs. 1392-1401). *
Munoz et al., 2008, Theriogenology, Vol. 69, pgs. 1159-1164. *
Paris et al., 2010, Theriogenology, Vol. 74, pgs. 516-524. *
Synnergren et al. (2010, Stem Cells and Develop., Vol. 19(7), pgs. 961-978). *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021099532A1 (en) 2019-11-22 2021-05-27 Novo Nordisk A/S Spin-aggregated neural microspheres and the application thereof

Also Published As

Publication number Publication date
EP2723852B1 (en) 2021-11-17
US10487313B2 (en) 2019-11-26
ES2902650T3 (es) 2022-03-29
US20200190477A1 (en) 2020-06-18
US20180002668A1 (en) 2018-01-04
JP2014519832A (ja) 2014-08-21
JP6312591B2 (ja) 2018-04-18
EP2723852A1 (en) 2014-04-30
CN103890167A (zh) 2014-06-25
WO2012175633A1 (en) 2012-12-27
CN108220224A (zh) 2018-06-29

Similar Documents

Publication Publication Date Title
US10487313B2 (en) Efficient induction of definitive endoderm from pluripotent stem cells
CN102083965B (zh) 多潜能细胞
EP2859091B1 (en) Differentiation of human embryonic stem cells into pancreatic endocrine cells
CA2809305C (en) Differentiation of pluripotent stem cells
CA2694956C (en) Pluripotent stem cell differentiation by using human feeder cells
CN102105580B (zh) 多潜能细胞的处理
RU2668814C2 (ru) Способы продуцирования клеток дефинитивной энтодермы и панкреатической энтодермы
HK1209160B (en) Differentiation of human embryonic stem cells into pancreatic endocrine cells

Legal Events

Date Code Title Description
AS Assignment

Owner name: CELLARTIS AB, SWEDEN

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:FUNA, NINA;HESS, KATJA;EKBERG, JENNY;AND OTHERS;SIGNING DATES FROM 20140312 TO 20140402;REEL/FRAME:032618/0971

Owner name: NOVO NORDISK A/S, DENMARK

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:FUNA, NINA;HESS, KATJA;EKBERG, JENNY;AND OTHERS;SIGNING DATES FROM 20140312 TO 20140402;REEL/FRAME:032618/0971

AS Assignment

Owner name: TAKARA BIO EUROPE AB, SWEDEN

Free format text: CHANGE OF NAME;ASSIGNOR:CELLARTIS AB;REEL/FRAME:037128/0385

Effective date: 20140918

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION