US20130280804A1 - Differentiation of human pluripotent stem cells to multipotent neural crest cells - Google Patents

Differentiation of human pluripotent stem cells to multipotent neural crest cells Download PDF

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US20130280804A1
US20130280804A1 US13/977,387 US201113977387A US2013280804A1 US 20130280804 A1 US20130280804 A1 US 20130280804A1 US 201113977387 A US201113977387 A US 201113977387A US 2013280804 A1 US2013280804 A1 US 2013280804A1
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Stephen Dalton
Laura M. Menendez
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells

Definitions

  • the present invention relates to the differentiation of human pluripotent cells, including human pluripotent stems cells (including hiPSCs) to produce a self-renewing multipotent neural crest cell population in a single step method preferably without the requirement of isolating intermediate cells and without appreciable contamination (in certain preferred instances, virtually none) with Pax6+ neural progenitor cells in the population of p75+ Hnk1+ Ap2+ neural crest-like stem cells.
  • the neural crest stem cell population obtained can be clonally amplified and maintained for >25 passages (>100 days) while retaining the capacity to differentiate into peripheral neurons, smooth muscle cells and mesenchymal precursor cells.
  • Neural crest stem cells are a multipotent cell population arising at the neural plate border of neural ectoderm between the neural plate and the non-neural ectoderm during vertebrate embryogenesis (1, 2). As neural crest cells delaminate from the roof plate upon closing of the neural tube, they migrate throughout the body where they contribute to the peripheral nervous system, connective and skeletal cranial tissues, melanocytes and valves of the heart. Specification of ectoderm into neural, neural plate border and epidermal cells is directed by overlapping but distinct combinations of signaling molecules centering around Wnt, BMP and Fgf pathways (3-10).
  • NPCs neural progenitor cells
  • 11-13 a wide-range of neuronal sub-types
  • NPCs Efficient methods for generation of NPCs from human pluripotent cells have recently been made possible by use of specific inhibitors, such as Noggin and SB 431542, that function by blocking BMP and Activin A/Nodal signaling, respectively (12). Simultaneous inhibition of these pathways is sufficient to drive pluripotent cells in culture down the neuroectoderm pathway, generating a population of Pax6 + Sox 1 + Sox2 + NPCs that can assemble into neural rosettes as columnar epithelia. When isolated from neural rosettes in culture, NPCs can be amplified due to their self-renewing capacity and further differentiated into a wide range of neural cell types (13, 14).
  • specific inhibitors such as Noggin and SB 431542 that function by blocking BMP and Activin A/Nodal signaling, respectively (12). Simultaneous inhibition of these pathways is sufficient to drive pluripotent cells in culture down the neuroectoderm pathway, generating a population of Pax6 + Sox 1
  • Neural crest cells are typically found interspersed with neural rosettes in such cultures and can only be obtained as a highly enriched cell population by cell sorting techniques (15, 16).
  • Alternative methods for generating neural crest cells from pluripotent cells have been described, but these utilize co-culture on feeder layers (16, 17), are relatively inefficient and also require cell sorting to generate highly enriched populations.
  • These methods all involve complex, multistep procedures that yield relatively low yields of p75 + Hnk1 + neural crest.
  • the present invention relates to a method for producing a population of p75+ Hnk1+ Ap2+ multipotent neural crest-like cells from human pluriopotent cells, preferably human pluriopotent stem cells or human induced pluripotent cells in a single step without appreciable contamination with Pax6+ neural progenitor cells in the population of neural crest-like stem cells produced.
  • This approach which involves differentiation of pluripotent cells through WNT signaling promotion and Activin A/Smad pathway blockade and in addition, in particularly preferred embodiments, the inhibition of bone morphogenic protein (BMP), directs the unexpectedly efficient differentiation of pluripotent cells to the neural crest-like stem cells.
  • the method of the present invention provides for the efficient generation of self-renewing neural crest stem cells that greatly enhance their potential utility in disease modeling and regenerative medicine.
  • human pluripotent cells preferably human pluripotent stern cells
  • WNT signaling promoter which may include a GSK inhibitor as otherwise described herein or a Wnt protein as otherwise described herein
  • an effective amount of at least one agent which inhibits Activin A and similarly acting TGFbeta family members such as TGF beta and nodal (Activin A inhibitor or other suppressor of the Activin A/Nodal, “Activin A inhibitor” or “Activin A/Smad pathway inhibitor”
  • TGF beta and nodal Activin A inhibitor or other suppressor of the Activin A/Nodal, “Activin A inhibitor” or “Activin A/Smad pathway inhibitor”
  • time generally, about 6 to 20 days, about 7 to 18 days, about 8 to 17 days, about 9-15 days, about 10-15 days, about 11 to 15 days, about 12 to 14 days, about 13 to 15
  • contamination of p75+ Hnk1+ Ap2+ neural crest-like stem cells with Pax6+ neural progenitor cells amounts to no more than 10%, no more than 7.5% no more than 5%, no more than 2.5%, no more than 1%, less than 1%, less than 0.5%, less than 0.1%, less than 0.05% and in certain embodiments, undetectable levels of Pax6+ neural progenitor cells.
  • NCSCs neural crest-like stem cells
  • hESCs and hiPSCs pluripotent cells
  • Wnt signaling under conditions of low global Smad signaling.
  • Cultures arising under these conditions are comprised of highly-enriched NCSCs that are devoid of other contaminating neuroectoderm cell types.
  • NCSCs can be maintained over extended periods in culture while retaining developmental potential for being differentiated into peripheral neurons and mesenchymal progenitor cells which mesenchymal progenitor cells may be further differentiated into osteocytes, chondrocytes and adipocytes.
  • the preferred method of the present invention produces NCSCs directly from human pluripotent cells without the need for co-culture on feeder-layers (the process is preferably feeder cell free) or cell sorting to obtain a highly enriched population (i.e., a population at least about 90% NCSCs).
  • FIG. 1 shows that hESC (WA09) differentiation to neuroprogenitor cells is inhibited by Wnt signaling.
  • A Schematic summarizing differentiation of pluripotent cells into neural progenitor cells and neural crest cells together with markers for the two cell types.
  • B Treatment of hESCs with SB 431542 (20 ⁇ M) and Noggin (500 ng/ml) promotes differentiation into Pax6+ cells but concurrent treatment with BIO suppresses this and generates p75+ Pax6 ⁇ neural crest-like cells. Scale bar, 100 ⁇ m.
  • C Flow cytometry showing that Dickkopf (Dkk) decreases the p75bright population in NPC cultures generated by treatment with SB 431542 and Noggin.
  • D Real-time PCR data for Ap2 and Pax6 from p75dim or p75bright sorted cells (from C).
  • FIG. 2 shows that hESC differentiation to neural crest requires Wnt signaling and is antagonized by Activin A and BMP pathways.
  • A Flow cytometry analysis of WA09 hESCs treated as indicated for 15 days. Cells were analyzed by probing with antibodies for p75 and Hnk1. The % of double negative and positive cells are indicated in the bottom left and top right quadrants, respectively.
  • B Immunocytochemistry and bright field (bottom right panel) of WA09 hESCs cells treated with BIO and SB 431542 for 12 days. Cells were probed with antibodies as indicated; p75, Pax6, Ap2, Hnk1 and for DAPI (DNA). Micron bar, 100 ⁇ M.
  • C RT-PCR transcript analysis of hESCs and neural crest (passage 10) cells treated with BIO and SB 431542. Transcript levels were normalized to Gapdh control. Assays were performed in triplicate and shown as +/ ⁇ standard deviation.
  • D Schematic illustration of the signaling requirements for neural crest differentiation from hESCs cells.
  • FIG. 3 shows the peripheral neurons derived from neural crest stem cells.
  • BIO, SB 431542-treated NCSCs were differentiated to peripherin+ ⁇ -tubulin+ cells for 14 days after switching to N2-based neural differentiation media. Fixed cells were then probed with antibodies for peripherin and ⁇ -tubulin. DNA was detected by staining with DAPI. Micron bar, 100 ⁇ m.
  • FIG. 4 shows the differentiation of neural crest cells into mesenchymal progenitors.
  • A Schematic illustrating possible differentiation pathways for neural crest stem cells (NCSCs).
  • B Bright field view of mesenchymal cells generated from neural crest after treatment for 4 days with 10% FBS-containing media. Micron bar, 100 ⁇ m.
  • C Loss of p75 expression detected by flow cytometry as NCSCs are converted to mesenchymal cells.
  • D Flow cytometry analysis showing marker expression (blue) in NCSCs and mesenchymal cells. Red, isotype control. The % of positive cells for each antigen in each quadrant is shown.
  • FIG. 5 shows the differentiation of neural crest derived-mesenchymal cells.
  • A Schematic showing the lineages capable of being formed from neural crest-derived mesenchymal cells in culture.
  • B Bright field picture (left hand panel) after differentiation into calponin+ smooth muscle actin+ (SMA) smooth muscle cells.
  • C Oil red O-stained adipocytes and (right hand panel) a bright field image of adipocytes showing oil droplets.
  • D Osteocytes produced by differentiation of neural crest-derived mesenchymal cells, detected by staining with Alizarin Red and alkaline phosphatase (AP) staining.
  • E Differentiation of mesenchymal cells to chondrocytes, detected by staining with Alcian Blue. Micron bar, 100 ⁇ m.
  • FIG. 6 shows the in vivo migration and differentiation of WA09 hESC derived NCSC.
  • A DiO labeled cells at time of injection and (B) 48 hours later showing cell migration.
  • C-F Immunocytochemistry and bright field images of the same microscopic field 72 hours after injection showing cells that had incorporated into a cranial ganglion area and differentiated to peripheral neurons. Cells were probed with antibodies for peripherin and hNA and counterstained with DAPI (DNA). Micron bar, 50 ⁇ m.
  • G-J Images of the same microscopic field showing a cluster of human NCSC (hNA-positive) in the head mesenchyme adjacent to the neural tube. Many of the cells are also Tuj1-positive. Micron bar, 20 ⁇ m.
  • FIG. 7 shows WA09 hESCs that were cultured in defined media (-Activin A) supplemented with SB 431542 and Noggin for 14 days. Cells were then fixed and stained with antibodies for Pax6, Sox1 and Sox2. DNA was detected by staining with DAPI.
  • FIG. 8 shows a luciferase assay for ⁇ -catenin activity in hESCs.
  • BIO treatment of WA09 hESCs activates ⁇ -catenin signaling as shown by activation of the Top-Flash luciferase reporter. Data is expressed as the fold-increase in luciferase activity over untreated cells after normalization to the Fop-Flash control. All assays were performed in triplicate and represented as +/ ⁇ standard deviation.
  • B Top-Flash activity in hESCs grown in defined media treated with Wnt3a, Dkk1, Wnt3a and Dkk1 or BIO. Experiments were performed in triplicate and represented as +/ ⁇ standard deviation.
  • FIG. 9 shows that WA09 hESCs treated with SB 431542, Noggin and BIO down-regulate pluripotency markers Oct4 and Nanog. hESCs were treated with the 3 factors for 12 days then fixed and stained with antibodies for Oct4 and Nanog. DAPI was used to visualize DNA.
  • FIG. 10 shows a western blot analysis for detection of pSmad1,5,8.
  • hESCs were grown for 3-6 days in the presence of BIO and SB 431542 with Noggin alone, BMP4 alone (5-50 ng/ml).
  • BIO and SB 431542 with Noggin alone, BMP4 alone (5-50 ng/ml).
  • Whole cell lysates were then probed with antibodies for Smad1, Cdk2 (load control) or pSmad1,5,8.
  • FIG. 11 shows hiPSC differentiation to neural crest cells.
  • A-B Flow cytometry analysis of hiPSCs treated with BIO and SB431542 in defined media lacking Activin A for 12 days. Flow cytometry analysis was performed using isotype antibody controls (left hand panel) and p75/Hnk1 antibodies (right hand panel). The % of positive cells in bottom left and top right quadrants are indicated.
  • C Bright field picture of neural crest cells analyzed in (A). Micron bar, 100 ⁇ m.
  • D Cells as in (C) were analyzed by immunocytochemistry by probing with antibodies for Ap2, Sox2, p75, Pax6. DNA was visualized by staining with DAPI. Micron bar, 200 ⁇ m.
  • FIG. 12 (FIG. S 6 ). shows that hiPSC-derived neural crest cells differentiate into multiple lineages.
  • A Generation of peripheral neurons is shown by staining with antibodies for peripherin and ⁇ -tubulin.
  • B Mesenchymal stem cells generated from hiPSC-derived neural crest can further differentiate into chondrocytes and osteocytes, as shown by staining with Alcian Blue, Alizarin Red and alkaline phosphatase (AP). Micron bar, 200 ⁇ m.
  • Standard techniques for growing cells, separating cells, and where relevant, cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques are those known and commonly employed by those skilled in the art.
  • a number of standard techniques are described in Sambrook et al., 1989 Molecular Cloning, Second Edition, Cold Spring Harbor Laboratory, Plainview, N.Y.; Maniatis et al., 1982 Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, N.Y.; Wu (Ed.) 1993 Meth. Enzymol. 218, Part I; Wu (Ed.) 1979 Meth. Enzymol.
  • primary Pluripotent Stem Cells also “primate Pluripotent cells” of which “human Embryonic Stem Cells” or hESCs are a subset and preferred for use in the present invention, are derived from pre-embryonic, embryonic, or fetal tissue at any time after fertilization, and have the characteristic of being capable under appropriate conditions of producing progeny of several different cell types that are derivatives of all of the three germinal layers (endoderm, mesoderm and ectoderm), according to a standard art-accepted test, such as the ability to form teratomas in 8-12 week old SCID mice.
  • the term includes both established lines of stem cells of various kinds, and cells obtained from primary tissue that are pluripotent in the manner described.
  • pluripotent cells or pPS cells are embryonic cells of various types, especially including human embryonic stem cells (hESCs), described by Thomson et al. (Science 282: 1145, 1998); as well as embryonic stem cells from other primates, such as Rhesus stem cells (Thomson et al., Proc. Natl. Acad. Sci. USA 92: 7844, 1995). Other types of pluripotent cells are also included in the term.
  • Human Pluripotent Stem Cells include stem cells which may be obtained from human umbilical cord or placental blood as well as human placental tissue.
  • Any cells of primate origin that are capable of producing progeny that are derivatives of all three germinal layers are included, regardless of whether they were derived from embryonic tissue, fetal, or other sources.
  • the pPS ce+lls are preferably not derived from a malignant source. It is desirable (but not always necessary) that the cells be karyotypically normal.
  • pPS cell cultures are described as “undifferentiated” when a substantial proportion of stem cells and their derivatives in the population display morphological characteristics of undifferentiated cells, clearly distinguishing them from differentiated cells of embryo or adult origin. Undifferentiated pPS cells are easily recognized by those skilled in the art, and typically appear in the two dimensions of a microscopic view in colonies of cells with high nuclear/cytoplasmic ratios and prominent nucleoli. It is understood that colonies of undifferentiated cells in the population will often be surrounded by neighboring cells that are differentiated.
  • Pluripotent stem cells may express one or more of the stage-specific embryonic antigens (SSEA) 3 and 4, and markers detectable using antibodies designated Tra-1-60 and Tra-1-81 (Thomson et al., Science 282:1145, 1998). Differentiation of pluripotent stem cells in vitro results in the loss of S SEA-4, Tra-1-60, and Tra-1-81 expression (if present) and increased expression of SSEA-1.
  • SSEA stage-specific embryonic antigens
  • Undifferentiated pluripotent stem cells typically have alkaline phosphatase activity, which can be detected by fixing the cells with 4% paraformaldehyde, and then developing with Vector Red as a substrate, as described by the manufacturer (Vector Laboratories, Burlingame Calif.) Undifferentiated pluripotent stem cells also typically express Oct-4 and TERT, as detected by RT-PCR.
  • pluripotent stem cells Another desirable phenotype of propagated pluripotent stem cells is a potential to differentiate into cells of all three germinal layers: endoderm, mesoderm, and ectoderm tissues.
  • Pluripotency of pluripotent stem cells can be confirmed, for example, by injecting cells into severe combined immunodeficient (SCID) mice, fixing the teratomas that form using 4% paraformaldehyde, and then examining them histologically for evidence of cell types from the three germ layers.
  • SCID severe combined immunodeficient
  • pluripotency may be determined by the creation of embryoid bodies and assessing the embryoid bodies for the presence of markers associated with the three germinal layers.
  • Propagated pluripotent stem cell lines may be karyotyped using a standard G-banding technique and compared to published karyotypes of the corresponding primate species. It is desirable to obtain cells that have a “normal karyotype,” which means that the cells are euploid, wherein all human chromosomes are present and not noticeably altered.
  • pluripotent stem cells include established lines of pluripotent cells derived from tissue formed after gestation, including pre-embryonic tissue (such as, for example, a blastocyst), embryonic tissue, or fetal tissue taken any time during gestation, typically but not necessarily before approximately 10-12 weeks gestation.
  • pre-embryonic tissue such as, for example, a blastocyst
  • embryonic tissue or fetal tissue taken any time during gestation, typically but not necessarily before approximately 10-12 weeks gestation.
  • Non-limiting examples are established lines of human embryonic stem cells or human embryonic germ cells, such as, for example the human embryonic stem cell lines WA01, WA07, and WA099 (WiCell).
  • the compositions of this disclosure during the initial establishment or stabilization of such cells, in which case the source cells would be primary pluripotent cells taken directly from the source tissues.
  • mutant human embryonic stem cell lines such as, for example, BG01v (BresaGen, Athens, Ga.), as well as normal human embryonic stem cell lines such as WA01, WA07, WA09 (WiCell) and BG01, BG02 (BresaGen, Athens, Ga.).
  • Epiblast stem cells and induced pluripotent stem cells (iPSCs) including human induced pluripotent stems cells (hiPSCs) fall within the broad definition of pluripotent cells hereunder and in concept, the technology described in the present application could apply to these and other pluripotent cell types (ie, primate pluripotent cells) as set forth above.
  • EpiScs are isolated from early post-implantation stage embryos. They express Oct4 and are pluripotent. See, Tesar et al, Nature , Vol 448, p. 196 12 Jul. 2007.
  • iPS cells are made by dedifferentiating adult somatic cells back to a pluripotent state by retroviral transduction of four genes (c-myc, Klf4, Sox2, Oct4). See, Takahashi and Yamanaka, Cell 126, 663-676, Aug. 25, 2006.
  • Human embryonic stem cells may be prepared by methods which are described in the present invention as well as in the art as described for example, by Thomson et al. (U.S. Pat. No. 5,843,780 ; Science 282:1145, 1998 ; Curr. Top. Dev. Biol. 38:133 ff., 1998 ; Proc. Natl. Acad. Sci. U.S.A. 92:7844, 1995).
  • embryonic stem cell refers to pluripotent cells, preferably of primates, including humans, which are isolated from the blastocyst stage embryo.
  • Human embryonic stem cell refers to a stem cell from a human and are preferably used in aspects of the present invention which relate to human therapy or diagnosis. The following phenotypic markers are expressed by human embryonic stem cells:
  • iPSCs induced pluripotent cells
  • hiPSCs human induced pluripotent cells
  • differentiated is used to describe a process wherein an unspecialized (“uncommitted”) or less specialized cell (a pluripotent stem cell as otherwise disclosed herein, preferably, a human embryonic stem cell) acquires the features of a more specialized cell, in this case, p75+ Hnk1+ Ap2+ neural crest-like stem cells.
  • a differentiated or differentiation-induced cell is one that has taken on a more specialized (“committed”) position within the lineage of a cell.
  • “De-differentiation” refers to the process by which a cell reverts to a less specialized (or committed) position within the lineage of a cell.
  • the lineage of a cell defines the heredity of the cell, i.e., which cells it came from and what cells it can give rise to.
  • the lineage of a cell places the cell within a hereditary scheme of development and differentiation.
  • a lineage-specific marker refers to a characteristic specifically associated with the phenotype of cells of a lineage of interest and can be used to assess the differentiation of an uncommitted cell to the lineage of interest.
  • the term “appreciable” within the context of contamination of p75+ Hnk1+ Ap2+ neural crest-like stem cells with Pax6+ neural progenitor cells means an amount which is less than about 10-15%, and preferably less than about 5-10% of the total amount or number of p75+ Hnk1+ Ap2+ neural crest-like stem cells which are produced using methods according to the present invention.
  • contamination of p75+ Hnk1+ Ap2+ neural crest-like stem cells with Pax6+ neural progenitor cells amounts to no more than 10%, no more than 7.5% no more than 5%, no more than 2.5%, no more than 1%, less than 1%, less than 0.5%, less than 0.1%, less than 0.05% and in certain embodiments, undetectable levels of Pax6+ neural progenitor cells.
  • the terms “differentiation medium”, “cell differentiation medium”, “culture media”, “basal cell medium”, “basal cell media” or “basal media” or “stabilizing medium” are used synonymously to describe a cellular growth medium in which (depending upon the additional components used) the pluripotent stem cells, preferably hESCs and/or neural crest cells as otherwise described herein) are produced, grown/cultured or alternatively and more particularly, differentiated into more mature cells.
  • Differentiation media are well known in the art and consist essentially of at least a minimum essential medium plus one or more optional components (in the case of the formation of neural crest cells, a defined media as known in the art, without Activin A in combination with the other components is preferably used as is disclosed herein).
  • Differentiation media for producing peripheral neurons include preferably neurotrophic factors) in effective amounts especially including for example, Heregulin ⁇ (at about 1-25 ng/ml, preferably about 10 ng/ml), BDNF (brain derived neurotrophic factor, about 5 to 25 ng/ml, preferably about 10 ng/ml), GDNF (glial cell line derived neutrophic factor, about 5-25 ng/ml, preferably about 10 ng/ml), neurotophin-3 (about 5-25 ng/ml, preferably about 10 ng/ml), NGF (nerve growth factor, about 5-25 ng/ml, preferably about 10 ng/ml), ascorbic acid (50-500 ⁇ M, preferably about 200 ⁇ M) and dbcAMP (Dibutyryl cyclic adenosine monophosphate, about 0.1 to about 1 mM, preferably about 0.5 mM), other optional components may include such components as LR-Igf (about 50-500 ng.
  • Preferred media includes Bovine Serum media (BS media) such as a basal cell media which may contain between 1% and 20% (preferably, about 2-10%) fetal calf serum, or for chemically defined medium (preferred) an absence of fetal calf serum and KSR, but including bovine serum albumin (about 1-5%, preferably about 2%).
  • BS media Bovine Serum media
  • Differentiation medium which is used is defined and preferably contains fetal bovine serum.
  • Preferred differentiation media is that which is described in the examples which follow.
  • the preferred media is a basal media or other chemically defined media as otherwise described which enhances Wnt signaling (using, for example, a GSK inhibitor or a Wnt protein as otherwise described herein) and inhibits the Activin A/Smad pathway using an Activin A inhibitor as otherwise described herein in an effective amount.
  • a BMP inhibitor as otherwise described herein is also included in the media.
  • inhibitors of Wnt signaling and promoters of Activin A and other promoters of the Activin A/Smad pathway such as Activin A, as well as BMP pathway promoters are preferably excluded from the differentiation media.
  • agents which optionally may be added to differentiation medium include, for example, nicotinamide, members of TGF- ⁇ family, including TGF- ⁇ 1, 2, and 3, Activin A, nodal, serum albumin, members of the fibroblast growth factor family, platelet-derived growth factor-AA, and -BB, platelet rich plasma, insulin growth factor (IGF-I, II), growth differentiation factor (GDF-5, -6, -8, -10, 11), glucagon like peptide-I and II (GLP-I and II), GLP-1 and GLP-2 mimetobody, Exendin-4, parathyroid hormone, insulin, progesterone, aprotinin, hydrocortisone, ethanolamine, epidermal growth factor (EGF), gastrin I and II, copper chelators such as, for example, triethylene pentamine, forskolin, Na-Butyrate, betacellulin, ITS, noggin, neurite growth factor, nod
  • suitable media may be made from the following components, such as, for example, Dulbecco's modified Eagle's medium (DMEM), Gibco #11965-092; Knockout Dulbecco's modified Eagle's medium (KO DMEM), Gibco #10829-018; Ham's F 12/50% DMEM basal medium; 200 mM L-glutamine, Gibco #15039-027; non-essential amino acid solution, Gibco 11140-050; ⁇ -mercaptoethanol, Sigma #M7522; human recombinant basic fibroblast growth factor (bFGF), Gibco #13256-029.
  • DMEM Dulbecco's modified Eagle's medium
  • KO DMEM Knockout Dulbecco's modified Eagle's medium
  • Ham's F 12/50% DMEM basal medium 200 mM L-glutamine, Gibco #15039-027; non-essential amino acid solution, Gibco 11140-050; ⁇ -mercaptoethanol, Sigma #M
  • a particularly preferred differentiation medium for growing/culturing pPSCs (especially, hESCs) and for differentiating cells in the present invention is defined media without Activin A (for neural crest differentiation) and DMEM/F12 (50:50) for peripheral neuron differentiation from multipotent neural crest cells which contains about 2% proalbumin (albumin; Millipore/Serologicals), 1 ⁇ Pen/Strep, 1 ⁇ NEAA, 1 ⁇ Trace Elements A, B, C (Mediatech), Ascorbic Acid (10-100 ng/ml, about 25-65 ng/ml, about 50 ng/ml), about 0.1 mM (0.025-0.5 mM) ⁇ -Mercaptoethanol (Gibco), about 2-10 ng/ml, about 5-9 ng/ml, about 8 ng/ml bFGF (Sigma), 200 ng/ml (5-500 ng/ml) LR-IGF (referred to as IGF-I; JRH Biosciences), and 10
  • Activin A is excluded. In other instances, depending upon the end product and the type of medium used (maintenance medium, etc.) Activin A is including at about 10 ng/ml (i.e., from about 1 ng/ml to no more than about 20 ng/ml).
  • Differentiation media useful in the present invention are commercially available and can be supplemented with commercially available components, available from Invitrogen Corp. (GIBCO), Cell Applications, Inc. and Biological Industries, Beth HaEmek, Israel, among numerous other commercial sources, including Calbiochem.
  • at least one differentiation agent such as fibroblast growth factor (FGF), LR-IGF (an analogue of insulin-like growth factor) and Heregulin (preferably all three in effective amounts) is added to the cell media in which a stem cell is cultured and differentiated into a neural crest cell, as otherwise described herein.
  • FGF fibroblast growth factor
  • LR-IGF an analogue of insulin-like growth factor
  • Heregulin preferably all three in effective amounts
  • the cell differentiation media comprises at least one promoter of Wnt Signaling such as a GSK inhibitor or Wnt protein as otherwise described herein alone, or optionally in combination with an inhibitor of Activin A/Smad pathway and further optionally in combination with a BMP inhibitor as otherwise described herein.
  • BMP inhibitors such as noggin, chordin, follistatin, sclerostin, gremlin, dorsomorphorin (dorsomorphin dihydrochloride), connective tissue growth factor (CTGF), among others, may also be used in conjunction with Activin A inhibitors and GSK inhibitors/Wnt proteins for the purpose of differentiation. A combination of these agents may be preferred.
  • Each of these components is used in any effective amount as otherwise described herein.
  • One of ordinary skill in the art will be able to readily modify the cell media to produce any one or more of the target cells pursuant to the present invention.
  • Cell differentiation medium is essentially synonymous with basal cell medium but is used within the context of a differentiation process and includes cell differentiation agents to differentiate cells into other cells, in the preferred methods, p75+ Hnk1+ Ap2+ neural crest-like stem cells.
  • Stabilizing medium is a basal cell medium which is used either before or after a differentiation step in order to stabilize a cell line for further use.
  • Culture media is essentially the same as stabilizing medium, but refers to media in which a pluripotent or other cell line is grown or cultured prior to differentiation.
  • cell differentiation medium and stabilizing medium may include essentially similar components of a basal cell medium, but are used within different contexts and may include slightly different components in order to effect the intended result of the use of the medium.
  • pluripotent stem cells also may be cultured on a layer of feeder cells that support the pluripotent stem cells in various ways which are described in the art, but in preferred embodiments the stem cells are differentiated in a feeder cell free environment.
  • pluripotent stem cells are cultured in a culture system that is essentially free of feeder cells, but nonetheless supports proliferation of pluripotent stein cells without undergoing substantial differentiation.
  • the growth of pluripotent stem cells in feeder-free culture without differentiation is preferred and is supported using a medium conditioned by culturing previously with another cell type.
  • the growth of pluripotent stem cells in feeder-free culture without differentiation is supported using a chemically defined medium.
  • GSK inhibitor is used to describe a compound which inhibits GSK (especially GSK3, including GSK3 ⁇ or GSK3 ⁇ ), and compounds which are GSK inhibitors are useful in promoting the WNT pathway pursuant to the present invention.
  • WNT pathway promoters which may be used in the present invention include GSK inhibitors as otherwise described herein.
  • preferred GSK inhibitors for use in the present invention include one or more of the following, all available from Calbiochem:
  • a preferred GSK inhibitor for use as a WNT pathway promoter in the present invention is BIO, described above, which may be used alone or in combination with another GSK inhibitor.
  • Other GSK inhibitors described may also be used, either alone or in combination with another GSK inhibitor and/or a Wnt protein, as described below, in further combination with an Activin A/Smad inhibitor (e.g., SB431542 as described below) and optionally, a BMP inhibitor.
  • Wnt proteins function similar to GSK inhibitors and in particular, GSK inhibitors according to the present invention. They are therefore subsumed under the term Wnt signaling promoter.
  • Exemplary Wnt proteins which may be used in the present invention include one or more of Wnt1, Wnt2, Wnt3, Wnt3a, Wnt4, Wnt10, Wnt14, Wnt14b, Wnt15, and Wnt16, among other Wnt proteins. The use of Wnt3a is preferred.
  • Wnt signaling promoters include GSK inhibitors such as BIO (GSK-3 IX) and Wnt3a, used alone or in combination.
  • the Wnt signaling promoter is used in combination with the Activin A/Smad pathway inhibitor and optionally, further in combination with a BMP inhibitor as otherwise described herein.
  • BIO and/or Wnt3a may be used alone, preferably in combination with the Activin A/Smad pathway inhibitor, in particular, SB431542 and further preferably, in combination with a BMP inhibitor, for example noggin, among others as otherwise disclosed herein.
  • Activin A inhibitor or “Activin A/Smad pathway inhibitor” is used generically to describe compounds or components which are added to a differentiation medium to inhibit the effects of Activin A/Smad pathway (thus inhibiting Activin A and other molecules which can signal through Smad such as TGFbeta, Nodal, and other compounds) in the differentiation process (Smad pathway inhibitor) and when used, produce neural crest-like stem cells from human pluripotent cells, including hESCs in high yield.
  • the differentiation agent comprises an effective amount of a GSK inhibitor and/or Wnt protein (preferably, a GSK3 inhibitor, such as BIO or other GSK3 inhibitor or a Wnt protein such as Wnt3a) and an Activin A inhibitor and optionally, a BMP inhibitor as otherwise described herein in a cell differentiation medium.
  • a GSK inhibitor and/or Wnt protein preferably, a GSK3 inhibitor, such as BIO or other GSK3 inhibitor or a Wnt protein such as Wnt3a
  • an Activin A inhibitor Activin A inhibitor and optionally, a BMP inhibitor as otherwise described herein in a cell differentiation medium.
  • Exemplary Activin A inhibitors for use in the present invention include, for example, SB-431542 (Sigma), follistatin (which also may be considered a BMP inhibitor in the present invention), follistatin gene related protein (FGRP, available from R and D Systems), BMP and Activin Membrane Bound Inhibitor (BAMBI), anti-BAMBI (monoclonal antibody), Smad7 (Mothers against Decapentaplegic Homolog 7) and TGF RI inhibitor (Calbiochern), among others.
  • Activin A inhibitors are used in the present invention in effective amounts, generally within the range of about 0.001 to about 100 ⁇ M or more, about 0.05 to about 75 ⁇ M, about 0.1 to about 50 ⁇ M, about 0.25 to about 35 ⁇ M, about 0.5 to about 25 ⁇ M.
  • the use of SB 431542 is preferred and is used preferably in combination with BIO and/or Wnt3a and an optional BMP inhibitor such as noggin.
  • BMP inhibitor is used to describe a compound which inhibits bone morphogenic protein signaling in a cell and facilitates the differentiation in effective amounts in combination with WNT Signaling Promoter and an Activin A/Smad Pathway Inhibitor.
  • Exemplary BMP inhibitors for use in the present invention include, for example, at least one inhibitor selected from the group consisting of noggin, chordin, follistatin (which also may be used as an Activin A/Smad pathway inhibitor), sclerostin, gremlin, dorsomorphorin (e.g. dorsomorphin dihydrochloride or StemoleculeTM) and connective tissue growth factor (CTGF), among others.
  • CGF connective tissue growth factor
  • BMP inhibitor a factor that block BMP signaling
  • BMP does inhibit neural cress formation if added. This is explained by low BMP signaling in the media which is presented in the examples (see Appendix A).
  • a BMP inhibitor in effective amounts represents a preferred embodiment of the present invention in a number of instances.
  • the term “activate” refers to an increase in expression of a marker such as p75, Hnk.1, Ap2 (in the case of the preferred neural crest-like stem cells) or Pax6 (in the case of neural progenitor cells) or an upregulation of the activity of p75, Hnk1, Ap2 or Pax6.
  • the term “isolated” refers to being substantially separated from the natural source of the cells such that the cell, cell line, cell culture, or population of cells are capable of being cultured in vitro.
  • the term “isolating” is used to refer to the physical selection of one or more cells out of a group of two or more cells, wherein the cells are selected based on cell morphology and/or the expression of various markers.
  • the term “express” refers to the transcription of a polynucleotide or translation of a polypeptide (including a marker) in a cell, such that levels of the molecule are measurably higher in or on a cell that expresses the molecule than they are in a cell that does not express the molecule.
  • Methods to measure the expression of a molecule are well known to those of ordinary skill in the art, and include without limitation, Northern blotting, RT-PCT, in situ hybridization, Western blotting, and immunostaining.
  • Markers describes nucleic acid or polypeptide molecules that are differentially expressed in a cell of interest.
  • differential expression means an increased level for a positive marker and a decreased level for a negative marker.
  • the detectable level of the marker nucleic acid or polypeptide is sufficiently higher or lower in the cells of interest compared to other cells, such that the cell of interest can be identified and distinguished from other cells using any of a variety of methods known in the art.
  • the term “contacting” i.e., contacting a cell with a compound
  • contacting is intended to include incubating the compound and the cell together in vitro (e.g., adding the compound to cells in culture).
  • the term “contacting” is not intended to include the in vivo exposure of cells to a differentiation agent that may occur naturally in a subject (i.e., exposure that may occur as a result of a natural physiological process).
  • the step of contacting the cell with differentiation medium and a Wnt signaling promoter e.g. a GSK inhibitor such as BIO or Wnt protein such as Wnt3a or as otherwise described herein
  • Activin A/Smad pathway inhibitor e.g.
  • the cells may be treated in adherent culture as an adherent layer, as embryoid bodies or in suspension culture, although the use of adherent layers may be preferred because they provide an efficient differentiation process oftentimes providing differentiation to a target cell population of 90% or more. It is understood that the cells contacted with the differentiation agent may be further treated with other cell differentiation environments to stabilize the cells, or to differentiate the cells further, for example to produce neural progenitor cells or other cells.
  • the cells are differentiated in a medium as otherwise disclosed herein comprising effective amounts of a Wnt signaling promoter (GSK inhibitor and/or Wnt protein as otherwise described herein in combination with an effective amount of an Activin A/Smad pathway inhibitor as otherwise disclosed herein and optionally, an effective amount of a BMP inhibitor.
  • a Wnt signaling promoter GSK inhibitor and/or Wnt protein as otherwise described herein in combination with an effective amount of an Activin A/Smad pathway inhibitor as otherwise disclosed herein and optionally, an effective amount of a BMP inhibitor.
  • BIO and/or Wnt3a in combination with SB431542 is preferred, as is the inclusion of a BMP inhibitor as otherwise described herein, in particular noggin.
  • Components which inhibit Wnt signaling and/or promote the Activin A/Smad pathway e.g.
  • Activin A are generally excluded or reduced in amount to have little or no impact on the result of the differentiation process.
  • the inclusion of a BMP inhibitor is not required (depending upon whether the differentiation medium enhances BMP activity, although in certain preferred instances, where it is desired to inhibit BMP signaling, a BMP inhibitor is also included in effective amounts.
  • differentiation agent refers to any compound or molecule that induces a cell such as hESC's, neural-like crest cells, neural progenitor cells, etc. to partially or terminally differentiate. While the differentiation agent may be as described below, the term is not limited thereto.
  • differentiation agent as used herein includes within its scope a natural or synthetic molecule or molecules which exhibit(s) similar biological activity.
  • an effective amount of a differentiation agent is that amount which, in combination with other components, in a differentiation medium will produce the differentiated cells desired.
  • the term “consisting essentially of” is used to describe media, in particular differentiation media which is used to differentiate pluripotent stem cells, especially human pluripotent stem cells such as human embryonic stem cells to neural-like crest cells which contains those components or elements which are useful for effecting the intended differentiation result (“the basic and novel characteristics of the present invention”) and limits or eliminates those elements which detract from providing the intended result of the present invention which is directed to a method for differentiating pluripotent cells to neural-like crest cells in high yield without appreciable contamination with Pax6+ neural progenitor cells.
  • WA09 WiCell
  • RUES1, RUES2 Dr. A. Brivanlou, Rockefeller University
  • hESCs and the hiPSC lines Fib2-iPS4 and Fib2-iPS5 were cultured on Geltrex (Invitrogen)-coated plates in chemically defined media containing Heregulin 13 (10 ng/ml), Activin A (10 ng/ml), LR-Igf (200 ng/ml) and Fgf2 (8 ng/ml) as described previously (25).
  • Neuroprogenitor Cell Neural Crest and Mesenchymal Cell Differentiation.
  • NPC differentiation was performed as described (18). Briefly, cells were plated on Geltrex-coated plates in defined media without Activin A, supplemented with 20 ⁇ M SB 431542 (Tocris) and 500 ng/ml Noggin (R&D systems) for 11 days with or without 2 ⁇ M of (2′Z,3′E)-6-Bromoindirubin-3′-oxime (BIO) (GSK3 Inhibitor IX, Calbiochem), or 15 ng/ml Dickkopf (R&D Systems). For direct neural crest differentiation, cells were plated at a density of 1 ⁇ 10 5 cells/cm 2 in defined media lacking Activin A, supplemented with 2 ⁇ M BIO and 20 ⁇ M SB 431542 (SB media).
  • Neural crest cells were cultured in media containing 10% fetal bovine serum and passed every 4-5 days.
  • Osteocyte, adipocyte and chondrocyte differentiation was performed according to manufacturer directions using StemPro Osteogenesis Kit, StemPro Chondrogenesis Differentiation Kit, StemPro Adipogenesis Differentiation Kit (Invitrogen), respectively.
  • Neural crest cells were passaged with Accutase then seeded on agarose-coated plates to form small-sized aggregates. Three days later cell aggregates were labeled with the fluorescent dye DiO as described (25). A slit was cut into HH stage 8-10 chick embryos in ovo at the junction of the non-neural ectoderm and the forming neural tube, and a small DiO labeled cell aggregate was inserted and positioned under a fluorescence stereomicroscope. Egg windows were sealed with transparent tape and eggs incubated for 48-72 h, after which embryos were evaluated for the presence of fluorescent cells.
  • Embryos were photographed to document the localization of DiO labeled cells, and then fixed in freshly prepared 4% paraformaldehyde overnight at 4° C. Embryos were rinsed three times in PBS, incubated in 30% sucrose/PBS overnight at 4° C., embedded in OCT and frozen in a bath of dry ice/isopentane. 8-12 ⁇ m sections were cut and processed for immunofluorescence detection of human nuclear antigen (hNA, Chemicon; 1:100), ⁇ -tubulin isotype III (Tuj1, Sigma: 1:400) or Peripherin (Santa Cruz Biotechnology, Inc: 1:200).
  • Sections were dried at room temperature for 10 min, rehydrated in PBS and permeabilized in PBS, 0.2% Tween 20 for 15 min. Sections were incubated with primary antibody diluted in 1% BSA/PBS, 0.1% Triton X-100 at 4° C. overnight in a humid chamber, washed three times for 10 min in PBS, 0.2% Tween 20, then incubated with a 1:200 dilution each of Cy2 conjugated goat anti-mouse IgG1 (hNA; Jackson Immunoresearch) and Cy3 conjugated goat anti-mouse IgG2b (Tuj1) or Donkey anti-Goat IgG (Peripherin) at 37° C. for 1.5 h.
  • Sections were washed twice in PBS, 0.2% Tween 20 and finally in PBS. Some sections were stained with DAPI (5 ⁇ g/ml) in PBS for 10 min prior to applying a coverslip using Prolong Gold (Invitrogen). Fluorescence was visualized and photographed on a Zeiss AXIO microscope.
  • Human pluripotent cells can be efficiently differentiated into Pax6 + NPCs by simultaneous inhibition of Activin A/Nodal and BMP signaling with SB 431542 and Noggin, respectively (see FIGS. 1A and B) (12).
  • Pax6 + Sox 1+ Sox2 + NPCs predominate in cultures where Smad signaling is blocked, relatively minor amounts of p75 + neural crest cells are also generated under these conditions ( FIGS. 1B and S 1 ), consistent with previous findings (12).
  • BIO (2′Z,3′E)-6-Bromoindirubin-3′-oxime
  • GSK3 glycogen synthase kinase 3
  • hESC-derived NCSCs could be maintained as a stable, self-renewing population over extended periods of culture in SBio-containing media (>25 consecutive passages). Similar results were obtained when different hESC lines and human induced pluripotent stem cells (hiPSCs) were treated with SBio-containing media (FIGS. S 6 and S 7 ). We conclude that activation of canonical Wnt signaling combined with low Smad2,3 and Smad1,5,8 activity are strict requirements for the efficient generation of neural crest-like cells from hESCs ( FIG. 2D ).
  • the neural crest is a multipotent population of cells arising from neural ectoderm in vertebrate embryos, capable of forming a diverse array of cell lineages.
  • FIG. 3A After culture in media containing a cocktail of factors (BDNF, GDNF, NGF, neurotrophin-3 and dbcAMP) that promote neural differentiation (18), ⁇ 75% of cells expressed ⁇ -tubulin and a similar number expressed peripherin/neurofilament 4 ( FIG. 3A ), indicative of peripheral neurons. Similar results were obtained with two other hESC lines (RUES1 and RUES2) and hiPSCs ( FIG. 3B-D ).
  • BDNF BDNF
  • GDNF GDNF
  • NGF neurotrophin-3
  • dbcAMP neurotrophin-3 and dbcAMP
  • Neural crest cells can also form mesenchymal precursor cells in vitro (15, 21). By culturing cells in media containing 10% FBS (21, 22), we confirmed that SBio-generated NCSC can be efficiently converted to a cell type with mesenchymal properties over a 4-day period ( FIG. 4A-B ).
  • Mesenchymal cells produced were highly enriched for mesenchymal stem cell (MSC) markers such as CD73, CD44, CD105, CD13 but lost expression of p75 ( FIG. 4C-D ).
  • MSC mesenchymal stem cell
  • hESC-derived mesenchymal cells could be converted into smooth muscle cells, chondrocytes, osteocytes and adipocytes ( FIG. 5 ).
  • neural crest-like cells generated by our highly efficient one-step method using small molecule compounds is capable of multi-lineage differentiation. This is comparable to the developmental potential of neural crest cells isolated from NPC cultures by FACS sorting reported previously (15, 18).
  • migrating cells were observed in 13.
  • fluorescently labeled cells were observed in the head and pharyngeal regions ( FIG. 6B ), including the cranial ganglion (FIG. 6 C-J). The identity of cells was confirmed by staining with the human-specific nuclear antigen antibody (hNA; FIG. 6E ).
  • hNA-positive cells for expression of the neural markers Tuj1 or peripherin.
  • Double hNA/Tuj1 positive cells were observed in small clusters throughout the head mesenchyme ( FIG. 6G-J ).
  • hNA/peripherin-positive cells were also found in the mesenchyme and incorporated into host cranial ganglia ( FIG. 6C-F ). The injected cells therefore migrate and differentiate into peripheral neurons in vivo, consistent with the expected characteristics of neural crest cells.
  • the rationale for our directed-differentiation approach is based on the known roles of canonical Wnt signaling in neural crest formation during vertebrate development (3-5).
  • the signaling conditions for neural crest progenitor specification from hESCs and hiPSCs involve inhibition of GSK3, an antagonist of Wnt signaling, and inhibition of Activin A/Smad signaling with SB 431542.
  • BMP4 antagonized neural crest specification but inhibitors such as Noggin were not required due to the low basal level of Smad1,5,8 signaling in our system.
  • the activation of Wnt signaling was sufficient to divert cells from a Pax6 + NPC fate to a p75 + neural crest fate, both of which require low levels of global Smad signaling.
  • Wnt therefore controls a molecular switch that determines differential ectoderm fates arising from human pluripotent cells in culture.
  • the present invention is directed to the first method for directed differentiation of human pluripotent cells towards a neural crest fate.
  • the method is highly-efficient, cost-effective and precludes formation of contaminating Pax6 + NPCs.
  • Removing the need for FACS-assisted purification now provides a platform from which the basic biology of neural crest can be better understood and better applied to disease modeling.
  • This new approach also establishes a new starting point for the generation of neural crest at scale for applications in tissue engineering and regenerative medicine applications.
  • NCs Neural crest stem cells
  • the process is inefficient and requires cell sorting to obtain a highly enriched population.
  • No specific method for directed differentiation of human pluripotent cells towards NC has been reported prior to the present invention. This severely restricts the utility of these cells as a model for disease and development and for more applied purposes such as cell therapy and tissue engineering.
  • small molecule compounds in a single-step method for the efficient generation of self-renewing neural crest-like stem cells (NCSCs) in chemically defined media. This approach is accomplished directly from human pluripotent cells without the need for co-culture on feeder-layers or cell sorting to obtain a highly-enriched population.
  • pluripotent cells are efficiently specified along the neuroectoderm lineage towards p75 + Hnk1 + Ap2 + neural crest-like cells, with little or no contamination with Pax6 + neural progenitors.
  • This cell population can be clonally amplified and maintained for >25 passages (>100 days) while retaining the capacity to differentiate into peripheral neurons, smooth muscle cells and mesenchymal precursor cells.
  • NCSC-derived mesenchymal precursors have the capacity for differentiation into osteocytes, chondrocytes and adipocytes.
  • the present inventors have developed methods for the efficient generation of self-renewing neural crest stem cells that greatly enhance their potential utility in disease modeling and regenerative medicine.

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US10053667B2 (en) 2018-08-21
WO2012091978A3 (fr) 2012-10-11
EP2658966A2 (fr) 2013-11-06
WO2012091978A2 (fr) 2012-07-05
US20190017020A1 (en) 2019-01-17

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