Patent Application
20250002867
The present application relates to a method for producing pluripotent stem cells from somatic cells, particularly, blood cells.
iPS cells, which are pluripotent stem cells produced by reprogramming somatic cells, can be differentiated into various cells and are expected to be used in regenerative medicine, such as medical transplantation. Methods for producing pluripotent stem cells using somatic cells from various origins are known.
Producing pluripotent stem cells from blood cells, particularly, peripheral blood mononuclear cells, is advantageous in that it is less invasive and less stressful for the patient. However, the reprogramming efficiency is quite low, and methods to improve the efficiency have been demanded. In addition, blood cells do not adhere to the bottom surface of a plastic petri dish or the like and can be advantageously grown in suspension culture. Blood cells also have an advantage in ease in handling in culture using an automatic culture apparatus or the like.
An object of the present application is to provide a method for producing pluripotent stem cells.
The present application provides the following embodiments.
The present application provides a method for producing pluripotent stem cells with high efficiency. Further, the present application provides a container, a system, a kit, and a program for producing pluripotent stem cells.
In the present specification and the claims, a numeric value with “about” is intended to include a ± 10% range of the value. For example, “about 20” includes “18 to 22.” A numerical range includes all numeric values between both end points and the numeric values of both end points. “About” for a range is applied to both end points of the range. Thus, for example, “about 20 to 30” includes “18 to 33.”
The present application provides a method for producing pluripotent stem cells from somatic cells, the method including the steps of
In the present disclosure, somatic cells are not particularly limited and thus any somatic cells can be used. Examples of somatic cells include epithelial cells to be cornified such as cornified epidermal cells; mucosal epithelial cells such as epithelial cells of a tongue epithelium; exocrine gland epithelial cells such as mammary gland cells; hormone-secreting cells, such as adrenal medullary cells; cells for metabolism and storage such as hepatocytes; luminal epithelial cells constituting an interface boundary such as type I alveolar cells; the luminal epithelial cells of an inner duct such as vascular endothelial cells; cells having cilia with transport capability such as airway epithelial cells; extracellular matrix-secreting cells such as fibroblasts; contractile cells such as smooth muscle cells; cells for blood and an immune system such as peripheral blood mononuclear cells, umbilical cord blood, T lymphocytes; sensory cells such as rod cells; autonomic nervous system neurons such as cholinergic neurons; supporting cells for sensory organs and peripheral neurons, such as companion cells; nerve cells and glial cells for a central nervous system, such as astrocytes; pigment cells such as retinal pigment epithelial cells; and the precursor cells thereof including tissue precursor cells. The degree of cell differentiation and the age of the animal from which the cells are collected are not particularly limited. Even if undifferentiated precursor cells including somatic stem cells or finally differentiated mature cells are used, the cells can similarly be used as the origin of somatic cells according to the present application. Examples of undifferentiated precursor cells include neural stem cells, hematopoietic progenitor cells, mesenchymal stem cells, and tissue stem cells or somatic stem cells such as dental pulp stem cells. In an embodiment, the somatic cells may be floating cells such as blood cells, for example, peripheral blood mononuclear cells, hematopoietic progenitor cells, and CD34-positive cells. The blood cells originate from, for example, peripheral blood or cord blood.
In the present application, animals as a source of somatic cells are not limited. For example, mammals such as mouse, rat, hamster, guinea pig, cow, horse, pig, sheep, monkey, orangutan, chimpanzee, dog, cat, bird, and human are used. Primates are preferred, and human is more preferred.
In the present application, “pluripotent stem cells” are cells having both capability of differentiating into various cells and proliferating. In a known method, somatic cells are reprogrammed to produce pluripotent stem cells by causing a factor called a reprogramming factor to act on the somatic cells.
In the present application, a culture medium may be prepared by properly adding required factors to a basal medium used for culturing animal cells. Examples of basal medium include IMDM, Medium 199, Eagle's Minimum Essential Medium (EMEM), αMEM, MEM Zinc Option, IMEM Zinc Option, Dulbecco's modified Eagle's Medium (DMEM), DMEM/F12, Ham's F12, RPMI 1640, Fischer's medium, and a mixture thereof. The basal medium may contain blood serum such as fetal bovine serum (FBS), or may be serum-free. The basal medium may contain, for example, one or more serum substitutes, such as albumin, insulin, transferrin, selenium, KnockOut Serum Replacement (KSR) (serum replacement during ES cell culture) (Invitrogen), N2 supplement (Invitrogen), B27 supplement (Invitrogen), a fatty acid, a collagen precursor, a trace element, 2-mercaptoethanol, and 3′-thiol glycerol or one or more substances including a lipid, an amino acid, L-glutamine, GlutaMAX (Invitrogen), a non-essential amino acid, a vitamin, a growth factor, a small molecule compound, an antibiotic such as streptomycin, penicillin, puromycin, mitomycin; an antioxidant, pyruvic acid, a buffering agent, a mineral, a cytokine, and an equivalent thereof. A commercially available culture medium may be used as the basal medium. For example, a medium such as StemSpan ACF (STEMCELL Technologies) or StemFit (R) AK03N medium (AJINOMOTO CO., INC.) is available.
In step (1), somatic cells are seeded in a container having at least one compartment configured to permit gathering two or more somatic cells.
“Gathering” or “gather” of somatic cells means a state where a somatic cell is in contact with at least one other somatic cell and a plurality of somatic cells are gathered in one place. A compartment that can gather two or more somatic cells is not particularly limited as long as two or more somatic cells can be gathered. For example, a compartment in which two or more somatic cells can be gathered at one place by a physical force, including a force generated by their own weight, may be used. Specifically, the two or more somatic cells in a compartment can be gathered by rotating, rocking, or shaking the container. A compartment in which an inclined surface extends from the opening to the bottom, which allows the cells to be gathered at the bottom by their own weight can also be employed. When a container having a compartment with an inclined surface extending from the opening to the bottom is used, the container may also be rotated, rocked or shaken so that the cells are gathered. Somatic cells may be gathered at a point (one or more points) or may be gathered linearly in the compartment.
In the present aspect, the container has one or more compartments, each is configured to permit gathering two or more somatic cells. The number of the compartments included in the container is not particularly limited and a person skilled in the art may appropriately determine the number. The container may have one, two, three, four, five, six, seven, eight, nine or ten compartments or one to one hundred thousand or more compartments. For example, if the container is a 24-well plate having 1200 compartments in each well, the number of compartments is 28,800. In one embodiment, the container has the two or more compartments.
In the present aspect, the compartment may have a low cell adhesion property. Examples of compartments with low cell adhesion property may include, but not limited to, a compartment in which no artificial treatment to enhance cell adhesion, such as coating with an extracellular matrix or the like is performed in a commercial cell culture container, and a compartment in which a treatment to suppress cell adhesion such as coating with polyhydroxyethyl methacrylate (poly-HEMA) or a polymer of 2-methacryloyloxyethyl is provided phosphorylcholine (Lipidure) but are not limited thereto.
The number of somatic cells seeded in step (1) may be appropriately determined by a person skilled in the art and is not particularly limited. For example, the number of cells may be determined such that most of the somatic cells can come into contact with at least one other somatic cell in step (3). When the somatic cells are gathered at a point in a compartment in step (3), the number of the cells may be 2 to 500 cells/compartment, 2 to 400 cells/compartment, or 3 to 300 cells/compartment. For example, the number of the cells may be 5 to 500 cells/compartment, 10 to 500 cells/compartment, 50 to 400 cells/compartment, or 100 to 300 cells/compartment, e.g., about 200 cells/compartment. When the somatic cells are linearly gathered in step (3), the number of the somatic cells to be seeded may be determined according to the length of the portion where somatic cells are gathered or the portion where somatic cells are likely to be gathered (also referred to as “linear portion”). The shape of the linear portion is not particularly limited and may be determined depending on the shape of the container. For example, the linear portion may be a straight line or a curved line. In one embodiment, somatic cells are seeded such that a theoretical value obtained by calculating the number of somatic cells accumulated on a cross section perpendicular to the linear portion is about one or more, about ten or more, about 100 or more, about 1,000 or more, or about 10,000 or more, for example, about one to 10,000 or about 100 to 1,000. The theoretical value is calculated by the following formula:
(the mean value of the diameters of the somatic cells seeded in a compartment)×(the number of the somatic cells seeded in the compartment)/(the length of the linear portion). For example, if the mean value of the diameters of the somatic cells is 10 μm and the length of the linear portion in the compartment is 10 cm, the number of the cells may be about 104 cells or more/compartment, about 105 cells or more/compartment, about 106 cells or more/compartment, about 107 cells or more/compartment, or about 108 cells or more/compartment. For example, the number of cells may be about 104 to 108 cells/compartment or 106 to 107 cells/compartment.
In step (2), a reprogramming factor is brought into contact with the somatic cells.
Reprogramming factor
In the present application, the reprogramming factor means a substance used alone or in cooperation with a plurality of factors to induce a differentiated state of certain cells to a less differentiated state. The reprogramming factors may include a factor necessary for nuclear reprogramming and an auxiliary factor (cofactor) for increasing the efficiency of nuclear reprogramming. The reprogramming factor may be, for example, a gene (DNA, RNA), a gene product (e.g., mRNA, miRNA, or protein), a small molecule compound, and a combination thereof.
When the reprogramming factor is a gene or a gene product thereof, the reprogramming factor is at least one selected from the group consisting of an Oct gene family gene, a Sox gene family gene, a Klf gene family gene, a Myc gene family gene, a Lin gene family gene, a Nanog gene, and a gene product thereof (WO 2007/69666, Japanese Patent No. 5696282, Science, 2007, 318:1917-1920). Among the genes, at least one selected from the group consisting of a gene of the Oct gene family, a gene of the Sox gene family, a gene of the Klf gene family, a gene of the Myc gene family, and a gene product thereof is particularly preferred.
Specific examples of the genes of these families and combinations thereof are listed below. Hereinafter, only the names of the genes are listed, and the use of their gene products is also included.
More specifically, the following combinations are listed but are not limited thereto. In the following combinations, Sox2 gene may be replaced by Sox1 gene, Sox3 gene, Sox15 gene, Sox17 gene, or Sox18 gene. Klf4 gene may be replaced by Klf1 gene, Klf2 gene, or Klf5 gene. c-Myc gene may be replaced by T58A (active variant) gene, N-Myc gene, or L-Myc gene.
In the combinations of (1) to (24), the Oct3/4 gene can be replaced with another gene of Oct gene family (e.g., Oct1A or Oct6). The Sox2 gene (or Sox1 gene, Sox3 gene, Sox15 gene, Sox17 gene, or Sox18 gene) can be replaced with another gene of Sox gene family (e.g., Sox7 gene). The Lin28 gene can be replaced with another gene of Lin gene family (e.g., an Lin28b gene).
In addition, combinations including all the constituent elements of any one of the combinations (1) to (24) and further including any other substances may also be included in the category of “reprogramming factor” in the present application, although the combinations do not correspond to the combinations (1) to (24). Under the condition that somatic cells to be reprogrammed internally express some of the constituent elements in any one of the combinations (1) to (24) at a level sufficiently high for reprogramming, a combination of only other constituent elements than the constituent elements may also be included in the category of “reprogramming factor” in the present application.
In addition to the reprogramming factors discussed above, one or more reprogramming factors selected from the group consisting of Fbx15 gene, ERas gene, ECAT15-2 gene, Tcl1 gene, and β-catenin gene may be combined and/or one or more reprogramming factors selected from the group consisting of ECAT1 gene, Esg1 gene, Dnmt3L gene, ECAT8 gene, Gdf3 gene, Myb12 gene, ECAT15-1 gene, Fthl17 gene, Sall4 gene, Rex1 gene, UTF1 gene, Stella gene, Stat3 gene, and Grb2 gene may also be combined. These combinations are specifically described in WO 2007/69666.
A preferable reprogramming factor is, for example, at least one selected from the group consisting of Oct3/4 gene, Sox2 gene, Klf4 gene, c-Myc gene (or L-Myc gene), Lin28 gene, Nanog gene, and a gene product thereof. Two or more genes are preferably selected and combined from the group consisting of Oct3/4 gene, Sox2 gene, Klf4 gene, c-Myc gene (or L-Myc gene), Lin28 gene, Nanog gene, and a gene product thereof, and three or more genes are more preferably selected and combined from the group. In particular, combinations of reprogramming factors to be preferably introduced may include at least (1) Oct3/4 gene or a gene product thereof, Sox2 gene or a gene product thereof, and Klf4 gene or a gene product thereof, (2) Oct3/4 gene or a gene product thereof, Sox2 gene or a gene product thereof, Klf4 gene or a gene product thereof, and c-Myc gene or a gene product thereof, (3) Oct3/4 gene or a gene product thereof, Sox2 gene or a gene product thereof, Klf4 gene or a gene product thereof, and L-Myc gene or a gene product thereof. Most of all, a combination of Oct3/4 gene or a gene product thereof, Sox2 gene or a gene product thereof, and Klf4 gene or a gene product thereof, and a combination of Oct3/4 gene or a gene product thereof, Sox2 gene or a gene product thereof, Klf4 gene or a gene product thereof, and L-Myc gene or a gene product thereof are preferable. Most preferably, a combination of Oct3/4 gene, Sox2 gene, and Klf4 gene, and a combination of Oct3/4 gene, Sox2 gene, Klf4 gene, and L-Myc gene are employed.
If the reprogramming factor is a gene or a gene product thereof, the origin of the gene is not particularly limited and any species may be selected according to the origin of the cells to be reprogrammed. Although human or another mammal such as mouse, rat, rabbit, pig, or a primate such as monkey may be selected, human is preferably selected.
cDNA sequence information of the reprogramming factors can be obtained from a known database. For example, an accession number in GenBank described in WO 2007/069666 may be referred to. Nanog gene is referred to as “ECAT4” in the publication.
Among the reprogramming factors, mouse and human cDNA sequence information of particularly preferable four genes (Oct3/4 gene, Sox2 gene, Klf4 gene, L-Myc gene) will be described below.
Gene name: Mouse/Human
cDNA of the reprogramming factors can be easily isolated from living cells as an origin of the sequence, by using a known method such as a PCR on the basis of the above cDNA sequence information or sequence information registered in a known database.
The sequences of the genes and the gene products of the reprogramming factors are not limited to the wild type. Any variations may be included if reprogramming of somatic cells can be induced. For example, a gene or mRNA encoding an amino acid sequence, in which one or a few e.g., three or less, five or less, ten or less, 15 or less, 20 or less, 25 or less of amino acids are added, deleted, substituted, and/or inserted to the above discussed reprogramming factor and capable of inducing reprogramming can be used in the present application. This also holds true for protein encoded by the gene or mRNA. If the biological activity (the ability to induce reprogramming) is maintained, variants of the above discussed polypeptides, such as polypeptides in which one to several residues (e.g., 2, 3, 4, 5, 6, 10, 15 or 20 residues) of amino acid at the N-terminus and/or the C-terminus are deleted or added, polypeptide in which one to several residues (e.g., 2, 3, 4, 5, 6, 10, 15 or 20 residues) of amino acid are substituted, and a gene or mRNA encoding the polypeptide may also be used. The variants may include a fragment, an analog, and a derivative of a natural protein and a protein fused with other polypeptides (e.g., foreign signal peptide or polypeptide with an added antigen fragment). In particular, the variants may include a polypeptide having a sequence obtained by substitution, deletion and/or addition of one or more amino acids of a wild-type amino acid sequence and having the same biological activity (e.g. activity for inducing reprogramming) as the wild-type protein. When a fragment of a wild-type protein is used, the fragment typically comprises a contiguous region which is 70% or more, preferably 80% or more or 85% or more, more preferably 90% or more, 95% or more or 98% of the wild-type polypeptide (mature type in the case of secretory protein).
The number of amino acid alterations is not particularly limited and is, for example, 30% or less of all amino acids of the natural mature polypeptide, preferably 25% or less, more preferably 20% or less, more preferably 15% or less, more preferably 10% or less, 5% or less, or 3% or less, and is, for example, 15 amino acids or less, preferably 10 amino acids or less, more preferably 8 amino acids or less, more preferably 5 amino acids or less, or more preferably 3 amino acids or less. When one or more amino acids are substituted, it is expected that the activity of the protein can be maintained by substitution with an amino acid having a side chain with the same property. Such substitution is referred to in this application as conservative substitution. Conservative substitution includes, for example, substitution between amino acids in any group including basic amino acid (e.g. lysine, arginine, histidine), acidic amino acid (e.g. aspartic acid, glutamic acid), uncharged non-polar amino acid (e.g, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar amino acid (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), branched amino acid (e.g. threonine, valine, isoleucine) and aromatic amino acid (e.g. tyrosine, phenylalanine, tryptophan, histidine).
The modified protein has high homology to the amino acid sequence of the wild-type protein. For example, high homology is an amino acid sequence having an identity of 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 93% or greater, 95% or greater, or 96% or greater. The identity of the amino acid sequence can be determined using, for example, a BLASTP program (Altschul, S. F. et al., J. Mol. Biol. 215:403-410, 1990). For example, the NCBI (National Center for Biotechnology Information) BLAST web site allows performing a search using a default parameter. (Altschul S. F. et al., Nature Genet. 3:266-272, 1993; Madden, T. L. et al., Meth. Enzymol. 266:131-141, 1996; Altschul S. F. et al., Nucleic Acids Res.25:3389-3402, 1997; Zhang J. & Madden T. L., Genome Res. 7:649-656, 1997). For example, using the blast2sequences program (Tatiana A et al., FEMS Microbiol Lett. 174:247-250, 1999), which compares two sequences, an alignment of the two sequences can be made to determine the identity of the sequences. A gap is treated in the same way as a mismatch. For example, the identity value for the entire amino acid sequence of the natural type cytokine (a form of a mature type after secretion) is calculated. Specifically, the ratio of the number of matching amino acids to the total number of amino acids of the wild-type protein (mature type in the case of secretory protein) is calculated.
In addition, a gene or mRNA can include a silent mutation without changing the encoded amino acid sequence. In an AT(U)-rich gene, five or more consecutive bases of A or T(U) are substituted by G or C without changing the encoded amino acid sequence, thereby stably achieving high expression of the gene.
In another embodiment, a reprogramming factor may be a small molecule compound (e.g., a combination of VPA, CHIR99021, 616452, tranylcypromine, forskolin, and DZNep) used to induce dedifferentiation of differentiated cells, or miRNA. If the reprogramming factor is a small molecule, the reprogramming factor can be added to a medium (Science 9 Aug. 2013: Vol. 341, Issue 6146, pp. 651-654).
Contact of Reprogramming Factor with Somatic Cells
In step (2), the reprogramming factor is brought into contact with the somatic cells. When the reprogramming factor is a gene, the reprogramming factor can be brought into contact with the somatic cells by introducing an expression vector encoding the reprogramming factor into the somatic cells according to a known method. The type of expression vector is not particularly limited, and a known expression vector may be used. Examples of the expression vector include an episomal vector, an artificial chromosome vector, a plasmid vector and a viral vector.
The episomal vector is a vector that can replicate autonomously outside of a chromosome. Specific means of using the episomal vector are disclosed in Yu et al., Science, 324, 797-801 (2009). For example, an episomal vector can be used having loxP sequences arranged in the same direction on the 5′ side and the 3′ side of a vector element necessary for replication of the episomal vector. The episomal vector can be replicated autonomously outside a chromosome and thus can provide stable expression in a host cell without being integrated into a genome. However, it is desirable to remove the vector quickly after iPS cell production. The vector element required for replication of the episomal vector is held between two loxP sequences, and Cre recombinase is induced to act on the vector element to excise the vector element so that the autonomous replication function of the episomal vector can be eliminated. Thus, the vector can be removed from the iPS cells at an early stage.
Examples of the episomal vector include a vector containing a sequence necessary for autonomous replication derived from EBV or SV40 or the like as a vector element. In particular, the vector element necessary for autonomous replication is an origin of replication and a gene encoding a protein which is coupled to the origin of replication to control replication. For example, the vector element is a replication origin oriP and an EBNA-1 gene in EBV or a replication origin ori and an SV40LT gene in SV40.
Examples of artificial chromosome vectors include a YAC (yeast artificial chromosome) vector, a BAC (bacterial artificial chromosome) vector, a PAC (P1-derived artificial chromosome) vector, and a HAC (human artificial chromosome) vector.
The plasmid vector is not particularly limited if the plasmid vector can be expressed in somatic cells to be introduced. If the somatic cells to be introduced are mammalian, a normally used vector can be used as the plasmid vector for expressing animal cells. Examples of the plasmid vector for expressing animal cells include pA1-11, pXT1, pRc/CMV, pRc/RSV, and pcDNAI/Neo.
Examples of the viral vector include a retrovirus (including lentivirus) vector, an adenoviral vector, an adeno-associated viral vector, a Sendai viral vector, a herpesviral vector, a vaccinia viral vector, a poxviral vector, a polioviral vector, a Sindbis viral vector, a rhabdoviral vector, a paramyxoviral vector, and an orthomyxoviral vector. In the present specification, the viral vector means a vector having genomic nucleic acid derived from the virus and capable of expressing a transgene by integration of the transgene into the nucleic acid.
A Sendai viral vector is the preferred viral vector. A Sendai virus is a mononegaviral virus belonging to Paramyxoviridae (Paramyxoviridae; including genera such as Paramyxovirus, Morbillivirus, Rubulavirus and Pnemovirus) which contains a minus strand RNA (an antisense strand with respect to a sense strand encoding a virus protein) as a genome. The Sendai viral vector is a chromosome-nonintegrated viral vector, and the vector is expressed in the cytoplasm. Thus, the transgene is not integrated into a host chromosome, allowing the vector to be safely removed from the introduced cell once the cells are reprogrammed.
The Sendai viral vector includes, in addition to infectious virus particles, a composite of a virus core, a virus genome and a virus protein, or a composite containing non-infectious virus particles or the like and capable of expressing a gene to be integrated by introduction into cells. For example, a ribonucleoprotein (a core portion of the virus, i.e. RNP) composed of a Sendai virus genome and Sendai virus protein (NP, P and L protein) to be coupled to the genome can express the transgene in cells by introduction into the cells (WO00/70055). The introduction into the cells can be performed by a general method, such as electroporation, lipofection or microinjection. Such a ribonucleoprotein (RNP) is also included in the Sendai viral vector.
Examples of a known method for introducing an expression vector into somatic cells include an infection method for a viral vector (e.g., a retroviral vector (Cell 2007 Nov. 30;131(5):861-72, a Sendai viral vector (Japanese Patent No. 5963309)) and a calcium phosphate method, a lipofection method, a RetroNectin method, or an electroporation method for a plasmid vector (Nat Methods.2011 May;8 (5):409-12).
When the reprogramming factor is mRNA or miRNA, the contact of the reprogramming factor with somatic cells is, for example, a gene transfer method using synthetic mRNA (e.g., a calcium phosphate method, a lipofection method (Cell Stem Cell. 2010 Nov. 5; 7(5): 618-630) or an electroporation method).
When the reprogramming factor is a protein, the reprogramming factor can be brought into contact with somatic cells, e.g., by a direct infusion method (e.g., a needle method, a lipofection method, or an electroporation method) of protein (e.g., a cell membrane permeable recombinant protein) (Cell Stem Cell, 4, May 8, 2009, 381-384).
Step (2) may be performed before or after step (1). In one embodiment, step (2) is performed after step (1). In another embodiment, step (2) is performed prior to step (1).
In one embodiment, the method of the present application includes the step of expanding culture of the somatic cells prior to step (2). The culture conditions may be appropriately determined by a person skilled in the art and are not particularly limited. For example, when the somatic cells are peripheral blood mononuclear cells (PBMC), CD34-positive cells may be expanded by culturing in CD34-positive cell culture medium. A known medium may be appropriately used as CD34-positive cell culture medium. For example, a culture medium containing IL-6, SCF, TPO, Flt-3L, IL-3, and G-CSF can be used.
The culture temperature is not limited, may be about 30 to 40° C., and for example, about 37° C. The culture is conducted in an atmosphere comprising CO2, O2, and N2. The CO2 concentration is about 0.05 to 15%, preferably about 3 to 7%, more preferably about 4 to 6%, and most preferably about 5%. The O2 concentration is about 0.05 to 100% and preferably 2 to 25%. The N2 concentration is about 0.05 to 100% and preferably 30 to 75%.
The period of expansion culture may be appropriately determined by a person skilled in the art and is not particularly limited. The period may be one to twenty days or three to ten days, for example four to seven days, during which the culture medium is replaced. In addition, the culture medium may be continuously replaced (perfusion culture) using, for example, an automatic culture apparatus.
In the expansion culture step, the somatic cells may be cultured by suspension culture. In the present application, “suspension culture” means that cells are cultured without adhering to a culture base material. Among commercially available cell culture containers, although not particularly limited, culture may be performed using a container in which no artificial treatment (e.g., coating with an extracellular matrix or the like) for improving adhesion with cells is performed or in which a treatment for artificially suppressing adhesion is performed (e.g., coating with polyhydroxyethyl methacrylate (poly-HEMA) or a polymer of 2-methacryloyloxyethyl phosphorylcholine (lipidure)). For example, commercial products such as 96-well low-adhesion plate (Sumitomo Bakelite Co., Ltd.) and 35-mm low-adhesion dish (Sumitomo Bakelite Co., Ltd.) may be used. If the somatic cells are floating somatic cells (e.g., blood cells such as peripheral blood mononuclear cells, hematopoietic progenitor cells, and CD34-positive cells), the floating somatic cells will not adhere to a culture vessel. Therefore, a culture container for adherent culture may also be used. For example, commercial products such as a 24F single well type adherent cell culture plate (Sumitomo Bakelite Co., Ltd.) may be used.
In step (3), the somatic cells in contact with the reprogramming factor are cultured in a state in which the two or more somatic cells are gathered in the compartment. The somatic cells may be cultured by suspension culture.
Step (3) is performed in a state where the two or more somatic cells are gathered in the compartment. For example, step (3) is performed in a state in which 2 to 500, 2 to 400, or 3 to 300 somatic cells may be gathered at a point in the compartment, and for example, 5 to 500, 10 to 500, 50 to 400, or 100 to 300 somatic cells, for example, about 200 somatic cells are gathered. If somatic cells are gathered in a linear portion in a compartment in step (3), the number of somatic cells to be gathered may be determined according to the length of the linear portion. In one embodiment, a theoretical value obtained by calculating the number of somatic cells accumulated on a cross section perpendicular to the linear portion is about one or more, about ten or more, about 100 or more, about 1,000 or more, or about 10,000 or more, for example, about one to 10,000 or about 100 to 1,000. Somatic cells may be gathered by actions such as rotating, rocking, and shaking the container, and somatic cells may be gathered at the bottom by using a compartment having an inclined surface extending from the opening to the bottom.
In step (3), the culture temperature is not limited, may be about 30 to 40° C., and for example, about 37° C. The culture is conducted in an atmosphere comprising CO2, O2, and N2. The CO2 concentration is about 0.05 to 15%, preferably about 3 to 7%, more preferably about 4 to 6%, and most preferably about 5%. The O2 concentration is about 0.05 to 100% and preferably 2 to 25%. The N2 concentration is about 0.05 to 100% and preferably 30 to 75%.
In step (3), the culture period of the somatic cells may be appropriately determined by a person skilled in the art based on, for example, the type of somatic cells, the type of reprogramming factor, the contact means of the reprogramming factor, and is not particularly limited. The culture period may be three to thirty days, five to twenty days, or eight to fifteen days, for example, about ten days. The generation of pluripotent stem cells can be confirmed by the expression of a pluripotent stem cell marker, e.g. SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49/6E, ALP, Sox2, E-cadherin, UTF-1, Oct3/4, Rexl and Nanog. Expression of pluripotent stem cell markers may be confirmed visually by immunostaining under a microscope, or by flow cytometry or fluorescence-activated cell sorting (FACS).
Produced pluripotent stem cells may be subcultured every three to thirty days, four to twenty days, or five to ten days, for example, every approximately seven days. The number of subculturing times is not particularly limited and may be, for example, one or more, two or more, three or more, four or more, five or more, ten or more, twenty or more, thirty or more, forty or more, fifty or more, sixty or more, seventy or more, eighty or more, ninety or more, or one hundred or more. The subculture period is not particularly limited and may be one day or more, ten days or more, twenty days or more, thirty days or more, forty days or more, fifty days or more, one hundred days or more, two hundred days or more, three hundred days or more, four hundred days or more, five hundred days or more, or 1000 days or more. For example, the container for suspension culture can be used as a container for subculture. When a closed automatic culture apparatus is used, subculture is not performed. The expansion and differentiation of the pluripotent stem cells may be performed by including the culture medium and/or refluxing the culture medium.
In one embodiment, in the method of the present application, the compartment includes a gathering portion that can gather two or more somatic cells with low cell adhesion and the step of gathering two or more somatic cells is further included. For example, the two or more somatic cells can be gathered by at least one following action; rotating, rocking or shaking the container. When the method of the present application includes the gathering step, the step (3) is performed after two or more somatic cells are gathered in a compartment by the gathering step. In other words, two or more somatic cells in contact with the reprogramming factor can be cultured in a state where the two or more somatic cells are more securely gathered.
Referring to
The container 100 of
The container 100 of
With reference to
The container 100 of
A container of
The present application provides a container comprising
The container of the present disclosure may have at least one compartment. For example, the container may be in the form of a plate, a package, or a dish.
The container of the present disclosure may further comprise a passage portion which allows the inside of the container body and the outside of the container body to communicate with each other and allows somatic cells to pass therethrough.
The guide portion is not necessarily pre-configured in a shape and may be a passageway or means that allows somatic cells to be guided to the collection portion by an external force.
A container 200 of
The container of
The container of
The container 200 of
The container 200 of
The container 200 of
The container 200 of
A container 300 of
For the recessed portion 310, any configuration that is capable of gathering two or more somatic cells that flow into the recessed portion 310 may be used. For example, as illustrated in
In
A container 400 of
The container 400 of
The passage portion 430 may be configured to perform all of the supply and discharge of somatic cells and the supply of reprogramming factors. Further, the passage portion 430 may be configured with a first passage portion that supplies and discharges somatic cells and a second passage portion that supplies the reprogramming factor or may be configured with a first passage portion that supplies somatic cells, a second passage portion that discharges somatic cells, and a third passage portion that supplies the reprogramming factor.
The shape and configuration of the recessed portion 421 of the container 400 are not limited to those of
The container of the present disclosure may contain the reprogramming factor in the compartment. The reprogramming factor may be used as the reprogramming factor in the compartment or may be the protein, a small molecule compound, or miRNA. The amount of the reprogramming factor contained in the compartment may be appropriately determined by a person skilled in the art and is not particularly limited.
The present application provides a system comprising:
As illustrated in
The container 2 may be a container having one or more compartments with low cell adhesion. For example, the containers 100, 200, 300, and 400 in
The controller 10 includes, for example, a CPU that performs operations and a memory device that stores programs necessary for the operations and results of the operations. In the system 1 according to the present embodiment, the controller 10 controls the reprogramming factor feeder 40, the tilting device 50, the shaking device 60, the rotating device 70, and the culturing device 80, in addition to the seeding device 20 and the collecting device 30, so that pluripotent stem cells are produced from somatic cells. The controller 10 monitors the operating conditions of various devices electrically connected to the controller 10. For example, when the container 100 in
The seeding device 20 supplies somatic cells to the container 2 before the somatic cells are brought into contact with the reprogramming factor, so that the somatic cells are seeded in the container 2. In the present embodiment, the seeding device 20 includes, for example, a storage tank in which somatic cells are stored before being brought into contact with the reprogramming factor, and a drive unit (e.g., a pump) that delivers somatic cells in the storage tank to the container 2.
The collecting device 30 discharges somatic cells in contact with the reprogramming factor from the inside of the container 2 to the outside of the container 2 and collects the somatic cells. In the present embodiment, the collecting device 30 includes, for example, a drive unit that discharges somatic cells in contact with the reprogramming factor from the inside of the container 2 to the outside of the container 2, and a storage tank that temporarily stores somatic cells discharged by the drive unit. Somatic cells stored in the storage tank are delivered by the drive unit to the culturing device 80.
The reprogramming factor feeder 40 comprises, for example, a storage tank in which the reprogramming factor is stored, and a drive unit which delivers the reprogramming factor in the storage tank to the container 2.
The tilting device 50 is configured to tilt a part of the container 2 or the entire container 2 with respect to a reference surface (e.g., the horizontal surface P in
The shaking device 60 is configured to rock or shake the container 2 so as to cause the two or more somatic cells in the container 2 to gather in the compartment of the container 2.
The rotating device 70 is configured to rotate the container 2 about the rotation axis L1 so as to cause the two or more somatic cells in the container to gather in the compartment by a centrifugal force.
The culturing device 80 cultures somatic cells in contact with the reprogramming factor, the somatic cells being collected by the collecting device 30.
The gathering-state monitoring device 90 includes, for example, an optical member such as a CCD (charge-coupled device) provided on a part of the rotation axis L1 or near the orbit of the container 2. The gathering-state monitoring device 90 is configured to monitor a gathering state of somatic cells when the container 100 is made of an optically visible material.
The system 1 may comprise five drive units for driving the seeding device 20, the collecting device 30, the reprogramming factor feeder 40, the tilting device 50, and the shaking device 60, respectively, or may comprise one to four drive units for driving some or all of the seeding device 20, the collecting device 30, the reprogramming factor feeder 40, the tilting device 50, and the shaking device 60.
The system of the present application may comprise an automated system for producing pluripotent stem cells from somatic cells. In this case, in addition to the foregoing devices, a device for more efficiently obtaining the automation may also be provided. When pluripotent stem cells are produced in accordance with GMP (Good Manufacturing Practice) standards, all of the devices constituting the system of the present application are preferably housed as an integrated system in a same dust-proof enclosure. In this case, however, the system of the present application may be configured to share some of the steps with other devices or allow an operator of the system of the present application to perform some of the steps. The devices constituting the system of the present application need not all be installed in the same facility. For example, only the controller 10 may be installed in a different facility and remotely connected to various devices such as the seeding device via the Internet.
The reprogramming factor feeder 40, the tilting device 50, the shaking device 60, the rotating device 70, the culturing device 80, and the gathering-state monitoring device 90 may be omitted. If the reprogramming factor feeder 40 is omitted, for example, the container 2 may be configured to include the reprogramming factor in the compartment in advance. If the tilting device 50 and the shaking device 60 are omitted, for example, the containers 200, 300, and 400 in
The present application provides a kit for producing pluripotent stem cells from somatic cells, the kit comprising
a container with low cell adhesion, wherein the container has one or more compartments each capable of gathering two or more somatic cells; and
a reprogramming factor. In this case, the kit may be configured such that the reprogramming factor is sealed in advance in the container or is sealed in another ampoule or the like. When a kit having the former configuration is provided, a kit user does not need to introduce another reprogramming factor after seeding somatic cells, and the somatic cells can be reprogrammed only by seeding the somatic cells in the container. Thus, pluripotent stem cells can be more easily produced under axenic conditions.
Examples of the container and the reprogramming factor that are used in the present aspect are configured as described above. The kit of the present application may further include a buffer solution, a culture medium, and an instruction manual or the like.
The present application further provides a program for causing a computer to perform the method for producing pluripotent stem cells from somatic cells that is disclosed in the present application. For example, the method for producing pluripotent stem cells from somatic cells is implemented by executing a predetermined program by the CPU of the controller 10 in the system 1 of
The program may be stored and delivered to the computer using various types of non-transitory computer-readable media. Non-transitory computer-readable media include, for example, a magnetic recording medium (e.g., a flexible disk, a magnetic tape, or a hard disk drive), a magneto-optical recording medium (e.g., a magneto-optical disk), a CD-ROM, a CD-R, a CD-R/W, and a semiconductor memory (e.g., a mask ROM, a PROM, a flash ROM, or a RAM). In addition, the program may be delivered to the computer over wired communication channels, such as an electric wire and an optical fiber or over a radio channel.
The present application provides a method for producing differentiated cells, the method comprising the steps of:
providing pluripotent stem cells produced by the method of the present application; and
culturing the provided cells in a culture medium for inducing cell differentiation.
The differentiated cells are not particularly limited but include muscular system cells such as a cardiac muscle cell and a skeletal myoblast, nervous system cells such as a neuron, oligodendrocyte, and a dopamine-producing cell, retinal cells such as a retinal pigment epithelial cell, hematopoietic system cells such as a blood cell and a myelocyte, immune cells such as a T cell, an NK cell, an NKT cell, a dendritic cell, and a B cell, cells constituting organs such as a hepatocyte, a pancreatic beta cell, and a nephrocyte, a chondrocyte, and gem cells or the like, and a progenitor cell or a somatic stem cell (e.g., a mesenchymal stem cell, a hematopoietic stem cell, and a neural stem cell) that is differentiated into these cells. In one embodiment, a differentiated cell is a cardiac muscle cell.
Induction of differentiation of pluripotent stem cells can be performed by any known technique. The conditions for differentiation are not particularly limited and may be appropriately adjusted according to the target differentiated cells. For example, the differentiation may be carried out by culturing pluripotent stem cells in a culture medium for inducing cell differentiation, wherein the culture medium for inducing cell differentiation is prepared by adding a predetermined cytokine, a growth factor, or other compounds of a predetermined concentration to a culture fluid. When the differentiated cells are cardiomyocytes, pluripotent stem cells may be subjected to induction of differentiation into cardiac muscle cells according to a method described in WO2021/172542.
The present application will be described in more detail below using examples. The present application is not limited by the examples. In the following examples, a day on which a reprogramming factor is brought into contact with somatic cells (if the contact lasts for several days, the starting date of the contact) is referred to as day 0, and the number of days that have elapsed is referred to as day X. Thus, for example, day 2 indicates two days after a day on which a reprogramming factor is brought into contact with somatic cells.
Table 1 shows the detail of reagent used in the present example.
A SRV™ iPSC-2 vector is a stealth RNA vector with a gene expression level optimized to produce iPS cells from a peripheral blood monocyte, a peripheral blood mononuclear cell, and a CD34-positive cell, and carries four human-derived reprogramming genes: a human OCT3/4 gene, a human KLF4 gene, a human SOX2 gene, and a human c-MYC gene, an EGFP gene derived from crystal jelly, and a puromycin (Puro) resistance gene derived from Streptomyces alboniger. The introduction of the SRV™ iPSC-2 vector into cells can be confirmed by observing fluorescence of EGFP under a fluorescence microscope. SRV™ iPSC-2 Vector is automatically deleted in response to the expression of miR-302, a micro RNA expressed specifically in pluripotent stem cells. SRV™ iPSC-4 Vector may be used.
AggreWell™ plate has 1200 microwells with a diameter of 400 μm in a well (see the right image in
Table 2 shows the detail of equipment used in the present example.
2. Origin cell
Table 3 shows the detail of cells (PBMC, ZenBio) used in the present example. Frozen PBMC was defrosted and used for the present example.
Immediately after seeding, cell colonies gathered at the bottom surface of the microwell of AggreWell™ were confirmed (
Expression of TRA-1-60, a pluripotent stem cell marker, was confirmed by immunostaining with TRA-1-60 antibody. On Day 10, TRA-1-60-positive cells were confirmed in almost all of the formed spheroids (the upper right image in
The result of example 1 is shown in Table 6. As shown in Table 6, PBMCs were aggregated at the beginning of the reprogramming step to efficiently obtain iPS cells.
Number of seeded cells: 1.2×104 cells/well
Reprograming period: 9 days
Reprograming reagent: TOKIWA-Bio inc., SRV™-iPSC-2 vector
To investigate the relationship between the size of a spheroid of cells to be formed first (the number of constituent cells) and reprogramming efficiency, the number of living cells, the number of TRA-1-positive cells, and the ratio of TRA-1-positive cells on Day 14 were measured with respect to the number of PBMC cells seeded for each microwell (1 to 500 cells/microwell). Results are shown in Table 7 and
The number of iPS cells in total was high when PBMCs were gathered at 300 cells/microwell. Efficiency was high when PBMCs were gathered at 5 to 100 cells/microwell.
According to the method described in example 1, iPS cell lines were established using a conventional method (scaffold dependent culture) and the method of the present application (suspension conditions). The number of PBMCs seeded for each microwell was 200. iPS cells were established using blood collected from three donors (n=3). The established iPS cells were collected and treated with fixative solution (4% PFA). The iPS cells were stained with SSEA4 antibody (Alexa Fluor (R) 647 mouse anti-SSEA-4, BD Biosciences) or Oct3/4 antibody (Oct-4A (C30A3) rabbit mAb, Cell Signaling Technology) and then were measured by flow cytometry (SA3800 Spectral Cell Analyzer, Sony Corporation). Membrane transport was performed before Oct3/4 antibody staining.
Table 8 shows the result of the mean value of iPS cells derived from three donors. From this result, it is found that the degree of undifferentiation of iPS cells established by the method of the present application was superior to that of the conventional method.
Reprogramming Efficiency When Cells Were Gathered Linearly
In examples 1 to 3, cells were seeded in the container (AggreWell™ plate) with a microwell shaped like an inverted pyramid. Thus, cells were gathered at the tip of one side (an inclined surface that is substantially triangular in shape) of the microwell that serves as a guide portion, that is, a point at the bottom. Therefore, the reprogramming efficiency in a state other than gathering to a point, i.e., linear gathering was examined.
The reprogramming factor was introduced into PBMCs by the same method as in example 1, and the cell suspension obtained in step B10 was seeded on a low-cell-adhesion culture plate having four microwells (compartments), each with a rectangular bottom surface measuring 80×27.9 mm. The number of cells seeded in four compartments were from 2.79×103 cells to 2.79×106 cells.
The culture plate was tilted approximately 45° after seeding, and cells were gathered linearly on one side of the bottom surface (short side) in each compartment and were cultured in this state. Table 9 shows the measurement result of the number of iPS cell colonies generated in each compartment on Day 14. “Accumulated cell number” in Table 9 is a theoretical value obtained by calculating the number of cells accumulated in each compartment in the vertical direction at the beginning of culture (Day 0) when the cells were 10 μm in diameter (example: 2.79×103 cells with a diameter of 10 μm were arranged horizontally in a line that measures 27.9 mm, and thus the number of accumulated cells is one in the compartment where 2.79×103 cells were seeded).
As shown in
Induction of Differentiation to Cardiomyocyte
The iPS cells (four lines derived from different colonies) produced by the method of example 1 were made into small pieces by using a 100-μM mesh and then were differentiation-induced to cardiomyocyte according to an established method (e.g., a method described in WO2021/172542). Specifically, after an embryoid was formed over three days, on the third day from the start of induction of differentiation, the culture medium was replaced with a StemPro-34 medium to which 1% of GlutaMAX (Invitrogen), 4×10−4 M monothioglycerol (MTG), 50 μg/ml of ascorbic acid (AA), 150 μg/ml of transferrin, 10 ng/ml of an vascular endothelial growth factor (VEGF; R&D Systems), 1 μM of IWP3 (Stemgent), 0.6 μm of dorsomorphin, and 5.4 μm of SB431542 were added, and the culture medium on the seventh day was replaced with 2 ml of a StemPro-34 medium to which 1% of GlutaMAX, 4×10−4 M MTG, 50 μg/ml of AA, 150 μg/ml of transferrin, and 5 ng/ml of VEGF were added. Thereafter, the culture medium was replaced with a culture medium having the same composition, every three to four days. From the start of induction of differentiation to the 12th day, cells were set in a hypoxic environment (5% O2), and then the cells were cultured in a normal oxygen environment (20% O2).
The cells were collected on the 15th day from the start of induction of differentiation, and the ratio of Troponin T-positive cells serving as a marker of cardiomyocyte was analyzed using a flow cytometer (using an anti-Troponin T antibody: Troponin T Ab-1(13-11) (Thermo Fisher Scientific, MS-295-P)). The ratio of troponin-positive cells in four lines was 6 to 30%.
Thus, it is found that differentiated cells (e.g., cardiomyocytes) are obtained by directly differentiation-inducing iPS cells produced by the method according to the present application.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/JP2021/035693 | Sep 2021 | WO | international |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2022/035937 | 9/27/2022 | WO |
