Patent Application
20150050733
Stem cells are characterized in their capability of proliferation while maintaining potency and differentiation capacity into specialized cell types. Such characteristics are also called stemness, which endows stem cells with great promise for scientific research and therapeutic applications. Considerable interests have been focused on cell stemness, especially how to reprogram somatic cells to acquire stemness. Previous work of Yamanaka et al. demonstrated that pluripotent stem cells can be generated from somatic cells via forced overexpression of key transcription factors, creating a new method for stemness manipulation (Takahashi and Yamanaka, 2006). Since then, great efforts have been devoted to designing safer and more efficient methods for induced pluripotent stem cells (iPS), such as the utilization of proteins (Kim et al., 2009; Zhou et al., 2009), RNAs (Warren et al.), microRNAs (Anokye-Danso et al.) or defined chemicals(Shi et al., 2008; Zhu et al.). Although these methods have proven effective in manipulating stemness of the cells, the efficiency and the risk control are still yet to be improved. Therefore, there exist great needs in finding alternative methods to reprogram cells.
One aspect of the present disclosure relates to methods of reprogramming cells comprising culturing the cells under a condition that allows formation of a three-dimensional (3D) cell aggregate, wherein the cells are induced into reprogramming.
In certain embodiments, the cells are cultured on a low-adherent substrate to allow formation of a three-dimensional cell aggregate. In certain embodiments, the low-adherent substrate comprises a hydrogel layer that is hydrophilic and neutrally charged. In certain embodiments, the low-adherent substrate comprises a hydrophobic surface.
In certain embodiments, the cells are cultured in suspension in a drop of culture medium hanging onto a supporting material to allow formation of a three-dimensional cell aggregate.
In certain embodiments, the three-dimensional cell aggregate is a multicellular and multilayer cell aggregate. In certain embodiments, the three-dimensional cell aggregate has a sphere-like shape.
In certain embodiments, the cells are reprogrammed to up-regulate one or more stem cell markers. In certain embodiments, the cells are reprogrammed to up-regulate one or more transdifferentiation markers.
In certain embodiments, the methods further comprising introducing an inducing agent into the culture. In certain embodiments, the inducing agent is a protein capable of inducing reprogramming of a cell. In certain embodiments, the inducing agent is a nucleic acid encoding for a protein that is capable of inducing reprogramming of a cell. In certain embodiments, the inducing agent is a chemical compound capable of inducing reprogramming of a cell.
Another aspect of the present disclosure relates to reprogrammed cells obtained using any of the methods provided in the present disclosure.
Another aspect of the present disclosure relates to reprogrammed three-dimensional cell aggregates obtained using any of the methods provided in the present disclosure.
Another aspect of the present disclosure relates to kits for reprogramming cells, comprising a low-adherent substrate, and optionally a culture medium.
A. Phase-contrast images of RT4 cells in monolayer (left) and sphere culture (right). Scale bars=200 μm.
B. Quantitative PCR (Q-PCR) analysis results for the stem cell marker genes of RT4 cells in monolayer and sphere culture.
C. Western blotting results for NANOG and SOX2 expression of RT4 cells in monolayer and 7-day-sphere culture. Teratoma cell line PA-1 was used as a positive control.
A. Quantitative analysis results of migrated cells in monolayer (left) and 7-day-sphere culture (right).
B Q-PCR analysis results for genes related with epithelial mesenchymal transition (EMT) of RT4 cells in monolayer and 7-day-sphere culture.
C. Table summarizing results of nude mice tumor formation assay of RT4 cells. Monolayer indicates trypsinized RT4 cells in monolayer culture; Sphere indicates trypsinized RT4 sphere cells.
A. Phase-contrast images of HEK293 cells in monolayer (left) and sphere culture (right). Scale bars=200 μm.
B. Q-PCR analysis results for the stem cell marker genes of HEK293 cells in monolayer and sphere culture.
C. Western blotting results for NANOG and SOX2 expression of HEK293 cells in monolayer and 10-day-sphere culture.
D. Luciferase assay indicating OCT4, NANOG and SOX2/OCT4 transcriptional activities in monolayer HEK293 and sphere HEK293 cells (n=3). Vector indicates control luciferase reporter. (M) indicates monolayer HEK293 cells and (S) indicates 10-day-sphere HEK293 cells.
E. Bisulfite genomic sequencing of the transcriptional regulatory regions of OCT4, NANOG and SOX2 of HEK293 cells in monolayer (panels on top row) and 10-day-sphere culture (panels on bottom row). The starting site is designated as +1. Open circles indicate unmethylated cytosine guanine dinucleotides (CpGs), while closed circles indicate methylated CpGs.
F. Q-PCR analysis results for embryonic stem (ES) cell marker genes of HEK293 cells in monolayer and sphere culture.
G. Q-PCR analysis results for marker genes of endoderm, mesoderm and ectoderm of HEK293 cells in monolayer and 10-day-sphere culture.
H. Alkaline phosphatase (AP) staining results for HEK293 cells in monolayer (left) and 10-day-sphere culture (right).
I. Table summarizing the results of nude mice tumor formation assay of HEK293 cells. “A” indicates trypsinized HEK293 cells in monolayer culture; “B” indicates trypsinized HEK293 cell spheres; “C” indicates non-trypsinized HEK293 spheres.
A. Sketch map of renal unit.
B-F. Q-PCR analysis results for genes expressed in metanephric mesenchyme (B), glomeruli (podocyte) (C), proximal tubule (D), Henle's loop (E), distal tubule and collecting duct (F) of HEK293 cells in monolayer, 5-day-sphere culture and 10-day-sphere culture.
A. Phase-contrast images of MEF cells in monolayer (left) and sphere culture (right). Scale bars=200 μm.
B. Q-PCR analysis results for the stem cell marker genes of MEF cells in monolayer and 7-day-sphere culture.
C. Q-PCR analysis results for the stem cell marker genes of MEF cells in monolayer and 12-day-sphere culture.
D. Western blotting analysis results for Sox2 expression of MEF cells in monolayer and 7-day-sphere culture.
E. Alkaline phosphatase staining results for MEF cells in monolayer (left) and 7-day-sphere culture (right).
A. Q-PCR analysis results for the marker genes of MEF cells in monolayer and 7-day-sphere culture.
B. Q-PCR analysis results for the marker genes of MEF cells in monolayer and 12-day-sphere culture.
C. Western blotting for Tuj1 and Gfap expression of MEF cells in monolayer and sphere culture. Mouse neurosphere was used as a positive control.
D-H. Percentage of cells positive for Tuj1 (D), Neurofilament (E), 5100 (F), Gfap (G) and GABA (H) in monolayer MEFs and in 7-day-sphere derived adherent MEFs.
I. Q-PCR analysis results of the marker genes of MEFs after neurogenic differentiation by butylated hydroxyanisole (BHA).
A-E. Percentage of transplanted MEFs in vivo positive for both GFP and neural markers including Tuj1 (A), GFAP (B), S100 (C), NeuN (D), or MAP2 (E).
A. Phase-contrast images of TTFs in monolayer (left) and sphere culture (right). Scale bars=200 μm.
B. Western blotting analysis results for Sox2 expression of TTFs in monolayer and 7-day-sphere culture.
C. Q-PCR analysis results for the marker genes of TTFs in monolayer and 7-day-sphere culture.
D-E. Percentage of cells positive for Tuj1 (D) and S100 (E) of Monolayer TTFs and 7-day-spheres derived adherent TTFs.
A. Phase-contrast images of MCF-7 cells in monolayer (left) and sphere culture (right).
B. Q-PCR analysis results for the marker genes of MCF-7 cells in monolayer and sphere culture.
A. Phase-contrast images of rat osteoblast cells in monolayer (left) and sphere culture (right).
B. Q-PCR analysis results for the marker genes of rat osteoblast cells in monolayer and sphere culture.
A. Phase-contrast images of rat neuronal stem cells in monolayer (left) and sphere culture (right).
B. Q-PCR analysis results for the marker genes of rat neuronal stem cells in monolayer and sphere culture.
The present disclosure provide methods for reprogramming cells under a condition that allows formation of a three-dimensional cell aggregate and induces the cells into reprogramming. The present disclosure also provides cells that are reprogrammed using the methods of the present disclosure. The present disclosure further provides compositions and kits for reprogramming cells using the methods of the present disclosure.
One aspect of the present disclosure relates to a method of reprogramming cells comprising culturing the cells under a condition that allows formation of a three-dimensional cell aggregate, wherein the cells are induced into reprogramming.
As used herein, the term “reprogram” or “reprogramming” refers to a process in which a differentiated cell is induced to generate one or more characteristics of a stem cell.
A differentiated cell is a cell that is committed to become or has become a specialized cell and shows one or more biological or functional characteristics of such specialized cell. A differentiated cell may be partially differentiated wherein the cell has not completed the process of becoming the specialized cell and does not contain all the characteristics of the specialized cell. A partially differentiated cell may also include a specialized cell that is in the process of reverting into a non-specialized cell and has lost some of the characteristics of the specialized cell. A differentiated cell may be terminally differentiated wherein the cell has completed the process of becoming the specialized cell and contain all the characteristics of the specialized cell. The term “differentiated cell” as used herein includes partially differentiated cells and/or terminally differentiated cells.
A stem cell is a cell that has the potential of differentiating into various specialized cell types. A stem cell may be pluripotent, i.e. capable of differentiating into most types of specialized cells, such as embryonic stem cells. A stem cell may be multipotent, i.e. capable of differentiating into certain types of specialized cells, such as bone marrow stem cells or hematopoietic stem cells. A stem cell may be unipotent, i.e. capable of differentiating into a specific type of specialized cell. A stem cell may be totipotent, i.e. a fertilized egg. The term “stem cell” as used herein includes any and all types of stem cells with the above mentioned various levels of potency.
The methods of the present disclosure may be used to reprogram any cells that can be induced to exhibit one or more biological and/or functional characteristics of stem cells under a culturing condition that allows formation of a three-dimensional cell aggregate. In certain embodiments, the cells of the present disclosure to be reprogrammed are adherent cells. Adherent cells are cells that require attachment to a solid substrate for growth in vitro (see, John M. Davis, Animal Cell Culture: Essential Methods., published by John Wiley & Sons in 2011, Section 4.2; Jennie P. Mather, et al., Introduction to Cell and Tissue Culture: Theory and Technique, published by Springer, 1998, p. 64-65). The adherent cells do not grow or live, or grow or live poorly in suspension cultures. Adherent cells are usually derived from solid tissues and organs. Examples of adherent cells include, without limitation, endothelial cells, smooth-muscle cells, epithelial cells and fibroblasts.
In certain embodiments, adherent cells has a less than 15% survival rate in suspension cultures. In certain embodiments, adherent cells has a less than 10% survival rate in suspension cultures. In certain embodiments, adherent cells has a less than 5% survival rate in suspension cultures. In certain embodiments, adherent cells has a less than 1% survival rate in suspension cultures. The cell survival rate is calculated as the percentage of the number of living cells out of the total number of cells in a cell culture. The cell viability may be measured by known methods such as propidium iodide staining.
In certain embodiments, the cells of the present disclosure to be reprogrammed by the methods of the present disclosure are somatic cells. Examples of somatic cells include, without limitation, fibroblast cells (e.g. mouse embryonic fibroblasts (MEFs), mouse tail-tip fibroblasts (TTFs) cells, and human fibroblast-like synoviocytes (HFLs)), urinary bladder cells (e.g., human urinary bladder papilloma (RT4) cells), baby hamster kidney (BHK21) cells, ovarian cells (e.g., Chinese hamster ovary (CHO-K1) cells), cervical cells (e.g. HeLa cells), muscle cells, embryonic cells (e.g. human embryonic kidney (HEK) 293 cells, NIH3T3 cells), lung cells (e.g. human fetal lung fibroblast (MRC-5) cells, MRC-9 cells), liver cells (e.g. Hep G2 cells), epithelial cells (e.g. WPE-stem cells), endothelium cells (e.g. HUV-EC-C cells), brain cells (e.g. T98G cells), bone cells (e.g. KHOS-2405 cells). In certain embodiments, the somatic cells are non-cancer cells.
In certain embodiments, the somatic cells are cancer cells such as breast cancer cells (e.g. MCF-7 cells, MDA-MB-231 cells, MDA-MB-435 cells or SK-BR-3 cells), colon cancer cells (e.g. DLD-1 cells, HCT-15 cells, T84 cells, HCT-8 cells), renal cancer cells (e.g. A-498 cells, 769-P cells, G401 cells), lung cancer cells (e.g. NCI-H2126 cells, DMS 79 cells, A549 cells), liver cancer cells (e.g., Hep G2 cells), cervical cancer cells (e.g. HeLa cells), glioma cells (e.g. M059K cells, LN-18 cells), neuronal cells (e.g., Rat pheochromocytoma (PC12) cells, Human neuoblastoma (SH-SY5Y) cells), prostate cancer cells (e.g., DU145 cells, LNCaP cells), and osteosarcoma cells (e.g. KHOS/NP cells).
In certain embodiments, the cells that are partially differentiated are induced into reprogramming by the methods of the present disclosure. In certain embodiments, stem cells are induced into reprogramming by the methods of the present disclosure.
In certain embodiments, the present disclosure provides a method of reprogramming cells including culturing the cells in vitro under a condition that allows formation of a three-dimensional cell aggregate, wherein the cells are induced into reprogramming. In certain embodiments, the culturing condition is a low-adherent condition. A low-adherent condition means a culturing environment in which the cells do not attach to a solid substrate or the cell attachment to the solid substrate is reduced or inhibited. In certain embodiment, in a low-adherent condition, cell attachment to a substrate is reduced by at least 30%, 40%, 50%, 60%, 70%, 80% or 90% when compared to a regular adherent condition. Regular adherent conditions may be provided by commercially available cell culture vessels, for example, Corning cell culture dishes (e.g. Cat. No. 430589 Corning® Not Treated Cell Culture Dish (New York, US) or Cat. No. 3353002 Falcon™ Cell Culture Dish (New York, US) or Cat. No. 715001 NEST Cell Culture Dish (Wuxi, China)).
In certain embodiments, the low-adherent condition is provided by a low-adherent substrate that reduces or inhibits cell attachment on its surface (see, John M. Davis, supra). In certain embodiments, the low-adherent substrates include cell culture vessels (e.g., a dish, plate, round-bottom tubes or flask) that are coated with a material that reduces or inhibits cell adhesion. In certain embodiments, such material contains a hydrogel layer that is hydrophilic and neutrally charged. A “hydrogel” as used herein, refers to a semisolid composition constituting a certain amount of water, and in which polymers or mixtures thereof are dissolved or dispersed. In certain embodiments, the hydrogel is made of polystyrene. In certain embodiments, the polystyrene is selected from the group consisting of polystyrene-block-poly(N-isopropylacrylamide)-block-poly styrene, poly(styrene-co-maleic anhydride) (SMA), poly(styrene-divinyl benzene) (P(ST-DVB)), poly(styrene sulfonic acid) (PSSA), poly(glycidyl methacrylate-co-α-methyl styrene), polyaniline-poly(styrene sulfonate) (Pan-PSS), poly(styrene-block-(methoxy diethylene glycol acrylate)-block-styrene) and poly(sodium 4-styrene sulfonate).
In certain embodiments, the hydrogel is made of agar, agarose. In certain embodiments, the low-adherent substrate is coated with agarose in a concentration ranging from 0.2 to 5.0%. In certain embodiments, the low-adherent substrate is coated with agar in a concentration ranging from 1-5%.
In certain embodiments, the hydrogel is made of poly(2-hydrozy-ethyl methacralate. In certain embodiments, the low-adherent substrate is coated with poly(2-hydrozy-ethyl methacralate in a concentration ranging from 40-50%.
In certain embodiments, the low-adherent substrate is coated with a hydrogel mixed with a culture medium (e.g., Dulbecco's Modified Eagle Medium (DMEM) or McCoy′5A Medium).
In certain embodiments, the material that reduces or inhibits cell adhesion can modulate the functions of extracellular proteins of a target cell and thus reduces attachment of the cell to the substrate. Examples of inhibitors of the extracellular proteins include, without limitation, proteoglycans (see, e.g. Yamagata, M. et al., Journal of Biological Chemistry, 264 (14): 8012-8018, 1989), heparin combined with hydrocortisone or cycloheximide, L-azetidine-2-carboxylic acid, trypsin and Ethylenediaminetetraacetic acid (EDTA), Dispase, dextran, polyethylene oxide, synthetic peptides such as glycine-arginine-glycine-asparate-serine-proline, glycine-arginine-glycine-asparate-serine (see, e.g. Whalen, G. F. et al., Ann. Surg., 210 (6): 758-764, 1989).
In certain embodiments, a material that reduces or inhibits cell adhesion is coated on the surface of the low-adherent substrate as a layer or film or membrane. In certain embodiments, a material that reduces or inhibits cell adhesion is embedded in the substrate.
In certain embodiments, the low-adherent substrate comprises a hydrophobic surface. In certain embodiments, a hydrophobic surface is formed with polymer and copolymer films having hydrophobic components. In certain embodiments, the hydrophobic surface is formed with poly(methyl methacrylate) (PMMA), poly(ethyleneoxide)-poly(methylmethacrylate) (PEO-PMMA), polydimethylsiloxane (PDMS), perfluoropolyether (PFPE), polyvinylidene fluoride (PVDF), polytetrafluoroethylene (PTFE), polystyrene (PS).
Any suitable culture medium may be used in a method of the present disclosure. Examples of commonly used culture medium include, without limitation, DMEM (Gibco®), RPMI 1640 (Gibco®), McCoy's 5A (Hyclone, Thermo Scientific), and DMEM/Nutrient Mixture F-12 (DMEM/F12, Gibco®).
In another aspect, the cells are cultured in suspension in a drop of culture medium hanging onto a supporting material such as the bottom of a petri dish and the wall of a rolling bottle. In certain embodiments, the drop of the culture medium has a volume of no more than 1 ml, 2 ml, 3 ml, 4 ml or 5 ml. In certain embodiments, the drop of culture medium hangs upside down on the bottom of a culturing container such as a petri dish.
In certain embodiments, the cells are cultured in a rotating, or shaking or static state.
In certain embodiments, the method of reprogramming cells comprises culturing the cells in the absence of any supporting substrate or structure suspending, floating or otherwise disposed within the culture medium.
The term “three-dimensional cell aggregate” as used herein refers to cells growing into a three-dimensional (3D) shape as opposed to a cell monolayer. A three-dimensional cell aggregate can be grown on two-dimensional, three-dimensional or multi-dimensional substrates. In certain embodiments, the three-dimensional cell aggregate of the present disclosure is a multicellular and multilayer cell aggregate. In certain embodiments, the three-dimensional cell aggregate of the present disclosure is in a sphere-like shape. In certain embodiments, the sphere-like shaped cell aggregate of the present disclosure can maintain a stable morphology for several weeks. In certain embodiments, the three-dimensional cell aggregate has a diameter of at least 10 μm, 15 μm or 20 μm.
In certain embodiments, the cells are reprogrammed to up-regulate one or more stem cell markers. The stem cell markers are genes and proteins that are characteristic of stem cells. Examples of stem cell markers for use with the methods described herein include, without limitation, Oct4, Nanog, Sox2, Klf4, c-Myc, Lin28, Rex1, Tdgf1, Leftb, Ebaf, Grb7, Podx1, Noda1, Fgf4, Nestin, Gdf3, Dax1, Nat1, Esg1, S1c2a3, Sox1, Olig2 and Pax6. The NCBI (National Center for Biotechnology Information) reference numbers of these stem cell markers (both nucleotide and protein) are shown in Table 1 below. The exemplary protein sequences are also shown in
In certain embodiments, the stem cell markers can be embryonic stem cells markers such as Oct4, Nanog, Sox2, Rex1, Gdf3, Dax1, Nat1, Esg1, S1c2a3, Tdgf1, Leftb, Ebaf, Grb7, Podx1, and Fgf4.
In certain embodiments, the stem cell markers can be renal stem cell markers such as Pax2, Wt1, Integrin Alpha 8, Sal11, Lim1, Ncam1, Six2, Frizzled 2, Frizzled 7, Acvr2b and Ntrk2.
In certain embodiments, the stem cell markers can be neural stem cell markers such as Sox2 (Episkopou, 2005; Reynolds and Weiss, 1992).
Reprogramming of a cell by a method of the present disclosure may be identified by the presence or absence of a stem cell marker or by the increase or decrease of a stem cell marker in the gene level or protein level. In certain embodiments, the cells that express no stem cell markers are induced into reprogramming by the methods of the present disclosure to express one or more stem cell markers. In certain embodiments, the cells that express one or more stem cell markers are induced into reprogramming by the methods of the present disclosure to express additional one or more stem cell markers. In certain embodiments, the reprogrammed cells of the present disclosure express at least two, three, four or five stem cell markers at the gene level and/or protein level.
In certain embodiments, the reprogrammed cells of the present disclosure express in an increased level of gene and/or protein of at least one, two, three, four, five or six stem cell markers selected from the group consisting of Oct4, Nanog, Sox2, Klf4, c-Myc, and Lin 28. In certain embodiments, the reprogrammed cells of the present disclosure express in an increased level of gene and/or protein of at least one, two, three, four or five stem cell markers selected from the group consisting of Oct4, Nanog, Sox2, Klf4, and c-Myc.
In certain embodiments, reprogramming of cells using the methods described herein cause cells to up-regulate stem cell markers Oct4, Sox2, c-Myc and Klf4 at the gene level while up-regulating stem cell markers Nanog and Sox2 at the protein level.
In another aspect, the methods of the present disclosure can also transdifferentiate a cell, in which a partially or terminally differentiated cell is transformed into a different type of differentiated cell, or an induced stem cell is transformed into a specialized cell that is different from the type of specialized cell before inducement.
In certain embodiments, the transdifferentiated cells are characterized by one or more transdifferentiation markers. The term “transdifferentiation marker” as used herein refers to characteristic gene and/or protein that can be used to identify transdifferentiation of a cell.
Examples of transdifferentiation markers may include, for example, endoderm markers such as Foxa2, Afp, Sox17 and Pdx-1, mesoderm markers such as Branchyury and Msx1, ectoderm markers such as Nestin, Otx2 and Tp63, renal glomeruli markers such as Nphs1, Actn4, Cd2ap, Cdh3, Pdpn and Podx1, renal proximal tubule markers such as Aqp1, Clcn5, Cubn, Lrp2 and Slc5a1, renal Henle's loop markers such as Umod and Pkd2, and renal distal tubule and collecting duct markers such as Scnn1a and Pkd1, neuron markers such as Map2, NeuN, Tuj1, NF-L, NF-M and NF-H, astrocyte markers such as Gfap, S100A and S100B, and oligodendrocyte markers such as Mbp and Ng2, and neuro-specific transcription factors such as Pax6, Sox1, Otx2, Zic1, NeuroD1 and Olig2. The NCBI reference numbers of the above mentioned transdifferentiation markers are shown in the Table 2 below. The exemplary protein sequences are shown in
In certain embodiments, cells reprogrammed using the methods of the present disclosure show both stem cell markers and transdifferentiation markers. For example, the methods of the present disclosure can induce an embryonic cell HEK293 to up-regulate certain stem cell markers (e.g., Six2, Frizzled2, Frizzled7, Acvr2b and Ntrk2) as well as certain transdifferentiation markers (e.g., Nphs1, Actn4, Cd2ap, Cdh3, Aqp1, Clcn5, Cubn, Umod Pkd2, Scnn1a, Pkd1), which indicates that the HEK293 cells have been transdifferentiated into mature renal cells.
In another aspect, the methods of the present disclosure further include introducing into the cell culture an inducing agent that is capable of inducing a differentiated cell to transform into a stem cell. An inducing agent can be any chemical or biochemical substance including, without limitation, a small molecule compound, a nucleotide, a peptide, or any combination thereof.
In certain embodiments, the inducing agent is a protein capable of inducing reprogramming of a cell. Examples of such proteins include, without limitation, Oct4, Sox2, c-Myc, Klf4, Nanog, and Lin28. The proteins can be recombinantly expressed in a suitable host cell and purified using methods known in the art (e.g. affinity chromatography). The proteins can be delivered to the cells using methods known in the art. For example, proteins can be delivered to cytosol by reversible permeabilization mediated by streptolysin O, which forms a pore in plasma membrane and allows protein delivery (see, e.g. Walev, I. et al, PNAS, 98(6): 3185-3190, 2001; Cho, H. J. et al., Blood, 116: 386-395, 2010). For another example, the proteins can be fused to a transduction domain such as HIV tat and poly-arginine, which can mediate the transmembrane delivery of the proteins to the cytosol (see, e.g. Zhou, H. et al., Cell stem cell, 4: 381-384, 2009).
In certain embodiments, the inducing agent is a polynucleotide encoding for a protein that is capable of inducing reprogramming of a cell. Examples of such polynucleotides include, without limitation, polynucleotides encoding for Oct4, Sox2, c-Myc, Klf4, Nanog, and Lin28. When introduced to the cells to be reprogrammed, the polynucleotides can be expressed to produce gene products that can stimulate the reprogramming. The polynucleotides can be an expression cassette containing the encoding sequence and promoter, or an expression vector, which can be readily constructed using molecular cloning techniques (e.g. cDNA preparation followed by insertion into a plasmid vector) and can be introduced into cells by transfection methods (e.g. using lipofectamine reagent or calcium phosphate).
In certain embodiments, the inducing agent is a chemical compound capable of inducing reprogramming of a cell. Examples of such chemical compounds include, without limitation, BIX-01294 and BayK8644 (see, e.g. Shi et al, cell stem cell, 3: 568-574, 2008), valproic acid, and 5′ azacytidine.
In another aspect, the present disclosure provides a reprogrammed cell obtained using the methods of the present disclosure. In certain embodiments, such reprogrammed cells show up-regulation in at least one, two, three, four, five or six stem cell markers. In certain embodiments, such reprogrammed cells show up-regulation in the gene and/or protein level of at least one, two, three, four, five or six stem cell markers selected from the group consisting of Oct4, Nanog, Sox2, Klf4, c-Myc and Lin28. In certain embodiments, such reprogrammed cells show up-regulation in at least one, two, three, four, five or six stem cell markers, and up-regulation in at least one, two, three, four, five or six transdifferentiation markers.
In another aspect, the present disclosure provides reprogrammed three-dimensional cell aggregates obtained using the methods of the present disclosure. In certain embodiments, the reprogrammed cell aggregates have sphere like shape. In certain embodiments, the reprogrammed cell aggregates have a diameter of at least 10 μm, 15 μm or 20 μm.
In another aspect, the present disclosure provides a kit for reprogramming cells, containing a low-adherent substrate and a culture medium. In certain embodiments, the kit further contains an instruction manual. In certain embodiments, the low-adherent substrate contains agarose or poly(2-hydrozy-ethyl methacrylate).
The reprogramming methods and the reprogrammed cells or cell aggregates of the present disclosure are useful for generating desired cells or tissues in vitro or in vivo. Such cells or tissues can be used for wound healing, neural re-generation, tissue re-generation, drug screening and the like.
All publications and patents cited in this specification are herein incorporated by reference to their entirety.
The invention will be more readily understood with reference to the following examples, which are not to be interpreted in any way as limiting the scope of the invention. All specific compositions, materials, and methods described below, in whole or in part, fall within the scope of the invention. These specific compositions, materials, and methods are not intended to limit the invention, but merely to illustrate specific embodiments falling within the scope of the invention. One skilled in the art may develop equivalent compositions, materials, and methods without the exercise of inventive capacity and without departing from the scope of the invention. It will be understood that many variations can be made in the procedures herein described while still remaining within the bounds of the invention. It is the intention of the inventors that such variations are included within the scope of the invention.
RT4 and Human Embryonic Kidney (HEK) 293 cells were cultured in McCOY′5A (HYCLONE) and DMEM (HYCLONE), respectively.
The mouse embryonic fibroblasts (MEF) cells were derived from E13.5 day ICR mouse embryos. For hippocampus transplantation experiment, green fluorescent protein (GFP) transgenic mouse was used. Briefly, after removing of head, limbs, visceral tissues and vertebral column, the remaining parts of the isolated embryos were cut into pieces followed by trypsinization. Cells were collected from the supernatant of the trypsinized tissues by centrifugation and then resuspended in fresh medium. The P0 MEF cells were allowed to grow to confluence before being passaged. The P2 MEF cells were use for further 2D and 3D culture.
Mouse tail tip fibroblast (TTF) cells were isolated from 24 h ICR mouse. Briefly, the front ¼-⅕ part of tail was cut into pieces which were then plated on to cell culture dishes and cultured for 2-3 days. The cells started to migrate out from the pieces at about 72 hours. After another 2-3 days culture, the cells migrating out were passaged. The P3 TTF cells were used for further 2D and 3D culture.
For 3D culture, low-adherent culture dish was prepared by coating cell culture dish with soft gel, which was made by mixing 1% melted agarose gel with an equal amount of 2×DMEM medium. 3×106RT4 or HEK293 cells were transferred to a 60 mm low-adherent dish. 6×106 MEF cells or TTF cells were transferred to 60 mm low-adherent dish.
For 2D culture, regular cell culture dish was used, without coating the dish with any soft gel.
Human urinary bladder papilloma cell line RT4 cells were cultured by monolayer adherent culture (2D) and sphere culture (3D), respectively. We used complete growth medium with 10% fetal bovine serum instead of serum-free medium supplemented with growth factors.
As shown in
Expression levels of the stem cell markers were determined using quantitative PCR (Q-PCR). Briefly, total RNA was isolated from the cells with Trizol LS reagent (Invitrogen), and then mixed with reverse transcriptase reaction mix (SuperScript III First-Strand Synthesis System, Invitrogen) and oligo-dT primers (Invitrogen). Q-PCR was performed using Power SYBR Green RT-PCR Kit (Applied Biosystems). GADPH was used as the control to normalize the cDNA inputs. The results were shown as fold change of the expression level of a testing marker in the testing cell culture relative to that in the 2D culture. Data were shown as mean±standard derivation, N=3. Student's T test was used to determine if the results for the 3D cultured cells are statistically different from the 2D cultured cells. Differences having statistical significance were marked with one asterisk (*, P<0.05) or two asterisks (**, P<0.01) in the Figures. All Q-PCR studies in the Examples and Figures were performed using the similar methods, unless otherwise specified.
Quantitative PCR analysis results of the genes expression showed that OCT4, NANOG, SOX2, KLF4 and c-MYC were up-regulated in sphere culture group compared to monolayer culture group (
The percentage of stem cells was detected by “Side Population” (SP) discrimination assay, a flow cytometry method for detection of stem cell percentage using dye efflux properties of stem cells. Briefly, cells were trypsinized and incubated with 5 ug/ml Hoechst 33342 (Sigma) at 37° C. for 90 min, either alone or with 50 ug/ml verapamil (Sigma). The cells were spun down and subject to flow cytometry cell sorting (FACS) analysis with a FACSVantage SE (BD Biosciences) after excluding the dead cells. The percentage of SP cells in sphere culture group was 0.83±0.32%, compared to 0.37±0.12% in monolayer culture group.
The metastasis ability of RT4 cell was detected by transwell assay, and the migrated cells was stained with crystal violet. Briefly, cells cultured by 2D or 3D method were serum-starved for 24 h before being trypsinized and resuspended in serum-free media. The cell suspensions were plated on the top chamber of a 24-well transwell containing 8 um pores (Corning), and the bottom chamber was filled with medium supplemented with 10% FBS. After 24 h, cells migrated in response to FBS to the bottom chamber were stained with 0.1% crystal violet, and the stained cells were counted under microscope. Quantitative analysis of the migrated RT4 cells indicated sphere culture promoted cell metastasis ability (
Gene expression associated with epithelial mesenchymal transition (EMT) was examined by Q-PCR. The results demonstrated FOXC2, SNAIL and ZEB2 were up-regulated in RT4 spheres compared to the monolayer (
We performed in vivo nude mice tumor formation assay. Cells were injected subcutaneously into the back flanks of 4-5 week old nude mice (8 BALB/c-nu). As shown in
HEK293 is a non-cancerous cell line originally derived from human embryonic kidney cells. HEK293 cells adhered to the cell culture dish showed monolayer epithelial morphology (
Expression of some stem cell markers was determined by Q-PCR and Western blotting using both the 2D cultured and the 3D cultured HEK293 cells. Q-PCR analyses of the known iPS inducing factors showed that OCT4, NANOG, SOX2, KLF4 and LIN28 were all up-regulated in sphere culture at 5 and 10 days compared to monolayer cultures (
The 2D cultured and the 3D cultured HEK293 cells were also compared for transcriptive activities of the stem cell markers, using reporter gene assay and bisulfite genomic sequencing.
For reporter gene assay, HEK293 cells from 2D or 3D cultures were transfected with luciferase reporter constructs of the binding sequences of OCT4, NANOG or SOX2/OCT4 (i.e., Oct4-Luc vector (constructed by inserting 6 W enhancer into PGL3 vector with tk promoter), Nanog-Luc vector (constructed by ligating Nanog binding site P5N to the SalI site of p37tk-luciferase vector, see also in: Pan, G. et al, Journal of Biological Chemistry, 280:1401-1407 (2005)), and Sox2/Oct4-Luc vector (constructed by cloning a sequence containing six copies of Oct4/Sox2 binding oligonucleotides to the upstream of the FGF-4 promoter in PGL3 vector). Luciferase assay was carried out according to the standard protocol (Promega). We found that OCT4, NANOG and SOX2/OCT4 proteins in 3D cultured cells had higher transcription activities (
For bisulfite genomic sequencing, bisulfite treatment was performed using Epi-Tect Bisulfite kit (Qiagen), and the transcription regulatory regions of OCT4, NANOG and SOX2 were amplified by PCR (Hotstar HiFidelity DNA polymerase) with respective primers shown in Table 3 below. The PCR products were subcloned into pMD18-T vector and 20 clones of each sample were picked out randomly and sequenced. Bisulfite genomic sequencing analyses evaluated the methylation status of cytosine guanine dinucleotides (CpG) in the transcriptional regulatory regions of OCT4, NANOG and SOX2, and the results revealed that the methylation status in HEK293 spheres were lower than those in monolayer HEK293 cells (
The 2D cultured and the 3D cultured HEK293 cells were further tested to see if they demonstrate any characteristics of undifferentiated ES cells. Q-PCR analyses of mRNA expression of several undifferentiated ES cell marker genes showed that REX1, TDGF1, LEFTB, EBAF, GRB7, PODX1 and FGF4 were up-regulated in HEK293 3D cells (
The tumorigenicity of HEK293 spheres were examined by in vivo nude mice tumor formation assay. 3×106 HEK293 monolayer cells and 3×106 HEK293 cells in sphere culture were injected subcutaneously into the back flanks of 4-5 week old nude mice, respectively. As shown in
In this example, HEK293 cells were tested for the reprogramming activities towards renal stem cell phenotype, and also the transdifferentiation reprogramming activities towards distinct types of terminally differentiated cells in kidney.
Kidney progenitor cells reside in the metanephric mesenchyme, which further differentiate into nephrons, which are composed of glomeruli (podocyte), proximal tubule, Henle's loop, distal tubule and collecting duct (
The 2D cultured and the 3D cultured HEK293 cells were tested for expression of metanephric mesenchyme specific genes and renal progenitor cell markers. Q-PCR analyses of the metanephric mesenchyme specific gene expression indicated that PAX2, WT1, INTEGRIN alpha 8, SALL1, LIM1 and NCAM1 were up-regulated in HEK293 spheres. The renal progenitor cell markers SIX2, FRIZZLED 2, FRIZZLED 7, ACVR2b and NTRK2 were also up-regulated in HEK293 spheres (
The 2D cultured and the 3D cultured HEK293 cells were further tested for nephron differentiation markers. The Q-PCR examination of differentiated markers of glomeruli, proximal tubule, Henle's loop, distal tubule and collecting duct showed that Glomeruli markers (NPHS1, ACTN4, CD2AP, CDH3, PDPN and PODXl), proximal tubule markers (AQP1, CLCN5, CUBN, LRP2 and SLC5A1), Henle's loop markers (UMOD and PKD2) and distal tubule and collecting duct markers (SCNN1a and PKD1) of HEK293 spheres were all up-regulated significantly in 3D cells when they were cultured for 5 and 10 days (
To further evaluate the effect of sphere culture on primary cell, MEFs were cultured into spheres. MEFs showed typical morphology with branched cytoplasm surrounding an elliptical nucleus when they were adhered to cell culture plates, while cultured on low-adherent plates they formed 3D spheres (
Considering the high expression of Sox2 in MEFs spheres, we tested whether MEF spheres acquired neurosphere properties. As illustrated in
When MEF spheres were re-adhered to cell culture dish, cells in the spheres migrated back onto the dish to form a monolayer. We name these back-migrated cells as sphere derived adherent cells. Immunocytochemistry staining was performed, and immunoflurescence pictures were taken for both monolayer cells and sphere cultured cells. For each cell culture, four representative pictures were taken, and percentage of cells that were positive for a particular marker was calculated for each picture by dividing the number of the positive cells by the total number of the cells in the picture. The results showed that some cells in sphere derived adherent cells were positive for Tuj1 (
Next we tested whether MEF spheres could acquire neural cell phenotypes in neural induction medium. Several methods were attempted including RA, SHH, N2 (data not shown) and BHA, only BHA was effective. After 10 days induction, monolayer MEFs showed no obvious morphology change, while cells derived from MEFs spheres exhibited long neural process-like morphology with extensive branching. Q-PCR results illustrated most neural markers of monolayer MEFs with BHA induction remained unchanged or down-regulated compared to control. In contrast, in MEF sphere group, most neural markers were further up-regulated including Sox2, Nestin, Map2, Gfap, Tuj1, Nf-L, S100B, Ng2, NeuN, NeuroD1 and Pax6 after BHA induction (
Hippocampus is a special structure in brain where neurogenesis occurs throughout adulthood. To further evaluate the neurosphere characteristics of MEFs spheres in vivo, GFP-labeled monolayer MEFs and sphere MEFs were transplanted into the hippocampus of rats, and the survival and differentiation capabilities were analyzed at 2 and 4 weeks. Compared to 2 weeks, the number of GFP positive cells at 4 weeks decreased dramatically in monolayer group while the number of sphere MEFs showed no apparent decrease, suggesting sphere MEFs possess higher viability. As shown in
Monolayer TTFs showed typical fibroblast morphology while TTFs cultured on low-adherent plates formed floating 3D spheres (
Human breast cancer cell line, MCF-7 cell line, was cultured in DMEM-High glucose medium (HYCLONE).
For 3D culture, low-adherent culture dish was prepared by coating cell culture dish with soft gel, which was made by mixing 1% melting agarose gel with an equal amount of 2×DMEM medium. 3×106 MCF-7 cells were transferred to a 60 mm low-adherent dish.
After a few days, the MCF-7 cells cultured in the 3D culture grew into spheres (see
The expression of several stem cell marker genes was examined by Q-PCR for both the MCF-7 monolayer formed in the 2D culture and the MCF-7 spheres formed in the 3D culture. As shown in
Rat osteoblast cells were cultured in DMEM-High glucose medium (HYCLONE). Rat osteoblast cells were obtained by primary isolation from 1-day old rat cranium.
For 3D culture, low-adherent culture dish was prepared by coating cell culture dish with soft gel, which was made by mixing 1% melting agarose gel with an equal amount of 2×DMEM medium. 3×106 rat osteoblast cells were transferred to a 60 mm low-adherent dish.
After a few days, the rat osteoblast cells cultured in the 3D culture grew into spheres (see
The expression of several stem cell marker genes was examined by Q-PCR for both the monolayer formed in the 2D culture and the spheres formed in the 3D culture. As shown in
Rat neuronal stem cells were cultured in DMEM/F12 (HYCLONE). Rat neuronal stem cells were obtained by primary isolation from 1-day old rat brain.
For 3D culture, low-adherent culture dish was prepared by coating cell culture dish with soft gel, which was made by mixing 1% melting agarose gel with an equal amount of 2×DMEM medium. 3×106 rat neuronal stem cells were transferred to a 60 mm low-adherent dish.
After a few days, the rat neuronal stem cells cultured in the 3D culture grew into spheres (see
The expression of several stem cell marker genes was examined by Q-PCR for both the monolayer formed in the 2D culture and the spheres formed in the 3D culture. As shown in
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/CN2012/072284 | 3/13/2012 | WO | 00 | 8/26/2014 |
