Patent Application
20210214692
This application contains a Sequence Listing, which was submitted in ASCII format via EFSWeb, and is hereby incorporated by reference in its entirety. The ASCII copy, created on Mar. 8, 2021, is named SequenceListing.txt and is 8 KB in size.
A stem cell can naturally divide or differentiate into another stem cell, progenitor, precursor, or somatic cell. However, sometimes a transient change in somatic cells can change its phenotype or express certain markers when placed in certain conditions. In this situation, the phenotype of many cells can be changed through forced expression of certain genes. However, once these factors are removed, the cells revert back to their original state. Therefore, these mechanisms of cell reprogramming have limited use.
True reprogramming was achieved with induced pluripotent stem cells (iPS cells or iPSCs) created independently by Yamanaka's group (71) and Thomson's group (72), although many of these cells were later found to be cancerous. These cells can be induced by true reprogramming since it was later shown that they can also be induced by non-gene integrating transient transfection (95, 96), by RNA (97) or protein (16, 98) or by small molecules (99). However, these cells are essentially identical to embryonic stem cells and have the same problems of uncontrolled growth, teratoma formation, and potential tumor formation.
Ideally, multipotent stem cells or pluripotent-like cells whose lineage and differentiation potential are more restricted can be developed so that they do not readily form teratomas or have uncontrolled growth. Thus, there is a need for methods of creating multipotent stem cells, multipotent stem-like cells, and stem-like cells and method of reprogramming or transforming easily obtainable cells to highly desirable multipotent stem cells, multipotent stem-like cells, and stem-like cells. This disclosure provides methods and materials for reprograming an easily obtainable cell into a cell that is generally difficult to obtain, or reprograming a vegetal cell to have new or different functionalities, without using stem cells.
In one aspect, provided is a method of reprogramming somatic cells into pluripotent stem cells. The method includes contacting one or more somatic cells with one or more antifolate agents in vitro for a period of time sufficient to induce reprogramming of the somatic cells, selecting one or more cells expressing one or more stem cell markers, growing the selected cells in a suitable medium to obtain the induced pluripotent stem cells (iPSCs). In certain embodiments, the method further comprises contacting the one or more somatic cells with one or more of glutamine, glutamate, arginine, methionine, and GABA. The method can further include growing the iPSCs in a suitable differentiation medium such that the selected cells differentiate into a desired lineage. In certain embodiments, the somatic cell includes but is not limited to a fibroblast cell and a stromal cell. In certain embodiments, the antifolate agent is a natural compound or a synthetic compound. In certain embodiments, the antifolate agent inhibits one or more C1 metabolites. In certain embodiments, the antifolate agent inhibits thymidylate synthase (TS), dihydrofolate reductase (DHFR), or both. In certain embodiments, the antifolate agent inhibits folypolyglutamate synthetase (FPGS). In certain embodiments, the antifolate agent includes but is not limited to methotrexate (MTX), pemetrexed (PTX), aminopterin (AMT), raltitrexed, trimetrexate, piritrexim, edatrexate, and fluorouracil. In certain embodiments, the induced pluripotent stem cell expresses one or more of the stem cell markers including OCT4, SOX2, SSEA-4, Nanog, and TRA 1-60. In certain embodiments, the somatic cell is contacted with the antifolate agent for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, such as 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or 15 days.
In another aspect, provided is an induced pluripotent stem cell (iPSC) obtained by reprogramming a somatic cell including but not limited to a fibroblast cell and a stromal cell. The obtained pluripotent stem cell expresses one or more of the stem cell markers including OCT4, SOX2, SSEA-4, Nanog, and TRA 1-60. In certain embodiments, the obtained pluripotent stem cell is capable of differentiating into 3 germ layers including mesoderm, endoderm, and ectoderm. In certain embodiments, the obtained pluripotent stem cell is capable of differentiating into a cardiomyocyte or a neuron. The reprogramming method includes contacting one or more somatic cells with one or more antifolate agents in vitro for a period of time sufficient to induce reprogramming of the somatic cell, selecting one or more cells expressing one or more stem cell markers, growing the selected cells in a suitable medium to obtain the induced pluripotent stem cells (iPSCs). In certain embodiments, the method further comprises contacting the one or more somatic cells with one or more of glutamine, glutamate, arginine, methionine, and GABA. The method can further include growing the iPSCs in a suitable differentiation medium such that the selected cells differentiate into a desired lineage. In certain embodiments, the antifolate agent is a natural compound or a synthetic compound. In certain embodiments, the antifolate agent inhibits one or more C1 metabolites. In certain embodiments, the antifolate agent inhibits thymidylate synthase (TS), dihydrofolate reductase (DHFR), or both. In certain embodiments, the antifolate agent inhibits folypolyglutamate synthetase (FPGS). In certain embodiments, the antifolate agent includes but is not limited to methotrexate (MTX), pemetrexed (PTX), aminopterin (AMT), raltitrexed, trimetrexate, piritrexim, edatrexate, and fluorouracil. In certain embodiments, the somatic cell is contacted with the antifolate agent for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, such as 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or 15 days.
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Disclosed herein are methods of reprogramming somatic cells into pluripotent stem cells by exposing the somatic cells to a high concentration of one or more antifolate agents such as MTX and PTX in vitro for an extended period of time sufficient for the somatic cells being reprogrammed into pluripotent stem cells, and selecting the pluripotent stem cells expressing one or more of the stem cell markers. In certain embodiments, the somatic cells are treated with variable concentrations of one or more of glutamine, glutamate, arginine, methionine, and GABA in addition to the one or more antifolate agents. In certain embodiments, both the antifolate agent(s) and the one or more of glutamine, glutamate, arginine, methionine, and GABA are added to the medium for growing the somatic cells. For example, the somatic cells are grown in a medium containing one or more of glutamine, glutamate, arginine, methionine, and GABA before and/or after treatment with the antifolate agent(s). In another example, the somatic cells are treated with the antifolate agent(s) while growing in a medium containing one or more of glutamine, glutamate, arginine, methionine, and GABA.
It was expected that at a high concentration, the antifolate agent would kill the cells or at least disrupt cell functions to a certain extent. Surprisingly, the inventors discovered that the treatment with the antifolate agents reprogrammed the somatic cell into an induced pluripotent stem cell rather than killing the somatic cell. The induced pluripotent stem cell expresses a number of stem cell markers, for example, one or more of the markers including OCT4, SOX2, SSEA-4, Nanog, and TRA 1-60, and is capable of differentiating into other types of cells such as cardiomyocytes and neurons.
Various concentrations of antifolate agents particularly methotrexate (MTX) and pemetrexed (PTX) were experimented in the cell culture medium. The standard practice to grow the normal/primary/somatic cells is 10% FBS-DMEM medium. As disclosed herein, various types and concentrations of FBS and different media formulations were tested to obtain the effective impact of MTX and/or PTX in less time. In some embodiments, the media formulations contain exogenous methionine at a range of between 0 and 30 mg/L such as about 1 mg/L, about 2 mg/L, about 3 mg/L, about 4 mg/L, about 5 mg/L, about 6 mg/L, about 7 mg/L, about 8 mg/L, about 9 mg/L, about 10 mg/L, about 11 mg/L, about 12 mg/L, about 13 mg/L, about 14 mg/L, about 15 mg/L, about 16 mg/L, about 17 mg/L, about 18 mg/L, about 19 mg/L, about 20 mg/L, about 21 mg/L, about 22 mg/L, about 23 mg/L, about 24 mg/L, about 25 mg/L, about 26 mg/L, about 27 mg/L, about 28 mg/L, about 29 mg/L, or about 30 mg/L, and the somatic cells were grown from 1 to 15 days such as 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or 15 days to induce reprogramming of somatic cells into iPSCs. Initially HEK 293T cells were used as the parental cells for the experiment. Once the positive results were obtained with the 293T cell, additional cells such as HF57 (human foreskin fibroblast) and DF (dermal fibroblast) cell lines were used as well.
To induce reprogramming of somatic cells into iPSCs, the parental somatic cells were grown in no methionine, low methionine (7.5 mg/L and 15 mg/L) to regular methionine (30 mg/L) DMEM+5% dialyzed-FBS supplemented with 1% glutamine, sodium hypoxanthine (10 mM) and thymidine (1.6 mM), and then treated for 1-15 days with one or more antifolate agents such as MTX, PTX, or both at various concentrations. For example, 293T cells were treated with 500 nM MTX and/or 1 μM PTX, and HF57 cells and DF cells were treated with 1 μM MTX and/or 1 μM PTX. Immunostaining of live cells with one or more stem cell markers such as SSEA4 (a pluripotentcy surface marker) was performed and the reprogrammed cells positively expressing the stem cell markers were selected using FACS. The independent cell colonies were selected and grown in an FBS-free stem cell medium such as MTeSR medium. These selected cells can be passaged to appropriate differentiation medium and grown into different lineages.
Pluripotent cells can differentiate into all cell types, so these cells can be a biological resource for regenerative medicine. Somatic cells can acquire the embryonic stem cell-like pluripotency by the introduction of four transcription factors, Oct4, Sox2, Klf4 and c-Myc (Yamanaka factors). Murine ES-like cell lines were established from mouse embryonic fibroblasts (MEFs) and skin fibroblasts by simply expressing these four transcription factor genes encoding Oct4, Sox2, Klf4, and c-Myc (71). After introducing active transcription factors into somatic cells, the somatic cells closely resemble ES cells in gene expression patterns, cell biologic and phenotypic characteristics. Since then, numerous studies have established that these transcription factors mediate reprogramming regardless of developmental origins and epigenetic states of a cell. These transcription factors are non-functional in somatic cells due to gene silencing. As demonstrated herein, the expression of Oct4, Sox2, Nanog, and SSEA4 was significantly higher in the MTX/PTX treated cells. Using the technology disclosed herein, nuclear reprogramming of somatic cells was induced possibly due to active Oct4, Sox2, Nanog, and other pluripotency genes, reduced DNA methylation, reduced histone methylation, and altered metabolism of certain biochemical factors (such as glutamine, glutamate, and GABA). This leads to the formation of embryoid bodies from single cells and differentiation into the mesoderm, endoderm, and neuronal ectoderm lineages with high efficiency in the differentiation medium. The 3-Germ Layer Immunocytochemistry Kit (ThermoFisher Scientific, USA), which analyzes spontaneously differentiated embryoid bodies derived from pluripotent stem cells for the presence of all 3-germ layers, was used to confirm the reprogramming. The assay detected widely accepted markers characteristic of the three embryonic germ layers: beta-III tubulin (TUJ1) for ectoderm, smooth muscle actin (SMA) for mesoderm, and alpha-fetoprotein (AFP) for endoderm. Thus, the presence of SMA (mesoderm) and AFP (endoderm) was confirmed using this kit as demonstrated in the working example. The ectoderm which differentiates to form the nervous system (spine, peripheral nerves, neurons, and brain) was not tested with this kit; however, the presence of neurons was confirmed by a different marker. A Human Neural Stem Cell Immunocytochemistry Kit (ThermoFisher Scientific, USA) which enables a convenient image-based analysis of common markers of human neural stem cells such as Nestin was used. The findings clearly show that neural lineages were obtained with high efficiency in the neural differentiation medium. Together, the disclosed methods result in the presence of all 3-germ layers in the MTX/PTX treated cells and these treated cells can differentiate into all cell types including beta cells.
Tetrahydrofolate (THF) and its derivatives, collectively referred to as folates, constitute a group of cofactors critical for several major metabolic pathways in the cell. These cofactors participate in the addition and removal of one-carbon (C1) units in a set of reactions commonly referred to as C1 metabolism (1). The products of these C1 transfer reactions include purines, thymidylate, methionine, S-adenosyl methionine (SAM), and pantothenate (vitamin B5), all of which are crucial for normal cell function (2, 3). The partitioning of carbon units into various cellular outputs involves the following 4 major pathways: the folate cycle, the methionine cycle, the transsulphuration pathway, and the transmethylation metabolic pathways (4-6) (
Epigenetic marking by DNA and histone methylation depends on SAM (9). These epigenetic marks are established during mammalian development (10, 11), and are essential for the maintenance of cell identity (12, 13). A large body of evidence suggests that either deficiency or excess of folate can modulate DNA methylation in a cell-, gene-, and site-specific manner, and imbalanced methylation can reinforce a plethora of health conditions ranging from cardiovascular disease to depression (14-19). Loss-of-function mutations in enzymes that are involved in the folate cycle, methionine cycle, transsulphuration pathway, and the transmethylation metabolic pathways can lead to growth defects both in animals and in humans, underscoring the role of C1 metabolism in modulating cell growth (20, 21). Various studies indicate that a steady pool of folate cofactors is essential for actively dividing cells and is required for normal growth and development (22-24). Although much progress has been made toward understanding the biochemistry of enzymes involved in folate metabolism (25), genetic evidence for the biologic roles of these enzymes is still limited.
One of the key enzymes in folate metabolism is folypolyglutamate synthetase (FPGS), which catalyzes ATP-dependent sequential conjugation of Glu residues to folate, forming folypolyglutamates. Polyglutamylation is essential for the retention of folates within cellular compartments because nonglutamylated or monoglutamylated folates can transport across the mitochondrial membrane in either direction (25). The polyglutamate chain lengths of the folates differ from 1 cell type to another and within different organelles of a given cell, but in most eukaryotic cells, the penta- and hexaglutamate forms predominate (1). In mammals, there is only 1gene for FPGS, but there are 2 isoforms that are independently required in the cytosol and in the mitochondrial matrix (25, 26). Mitochondria receive folates from the cytoplasm only in a reduced, monoglutamylate form (
In plants and cancer cells, mutation of FPGS changes the glutamylation status of the folates, and this alteration in polyglutamylated folates and associated compounds affects DNA methylation and releases chromatin silencing on a genome-wide scale (30-32). Polyglutamylated folates are better substrates for methylene THF reductase and methionine synthase, and both of these enzymes are involved in the generation of SAM (33, 34). In cancer cells, FPGS down-regulation by small interfering RNA reduces global DNA methylation and DNA methyltransferase (DNMT) activities (32, 35).
Although FPGS plays a central role in C1 metabolism, folate metabolism, and transmethylation pathways, it was unclear how an imbalance in these pathways caused by FPGS mutation would affect mammalian cell growth and differentiation. Therefore, a null mutant of FPGS was characterized in a mammalian cell, eliminating cytoplasmic and mitochondrial isoforms, and 4 splicing variants (36, 37). As disclosed herein, homozygous deletions of FPGS in the human embryonic kidney (HEK) 293T cell line were created using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). The FPGS knockout)(FPGSko) cell lines are viable, displaying stem-cell markers in cell culture, but proliferate extremely slowly with a tendency toward cardiogenesis and neurogenesis.
The following examples are provided to better illustrate the claimed invention and the embodiments described herein, and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of invention, and it is understood that such equivalent embodiments are to be included herein. Further, all references cited in the disclosure are hereby incorporated by reference in their entirety, as if fully set forth herein.
As demonstrated in the studies below, the elimination of both FPGS isoforms in 293T cells triggered epigenetic modifications, influenced gene expression, assisted cellular plasticity, and reduced cell proliferation. Moreover, the FPGSko cells are directed toward cardiac and neuronal lineages. A substantial reduction in global DNA methylation and noteworthy changes in gene expression related to C1 metabolism, cell division, DNA methylation, pluripotency, Glu metabolism, neurogenesis, and cardiogenesis were found. The expression levels of NANOG, octamer-binding transcription factor 4, and sex-determining region Y-box 2 levels were increased in the mutant, consistent with the transition to a stem cell-like state. Gene expression and metabolite data also indicate a major change in Glu and GABA metabolism. In the appropriate medium, FPGSko cells can differentiate to produce mainly cells with characteristics of either neural stem cells or cardiomyocytes.
Cell lines and production of FPGS mutants: HEK cell line 293T was cultured in DMEM (Corning, Corning, N.Y., USA) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, Mass., USA) and 1% GlutaMax (Thermo Fisher Scientific). The cell lines were cultured at 37° C. in a humidified 5% CO2 incubator. Stable clonal cell lines were created by transfecting 293T cells with GeneArt CRISPR Nuclease Vector with orange fluorescent protein (OFP) reporter gene (Thermo Fisher Scientific) (38) (
Plasmid and sgRNA design: The sgRNAs targeting the human FPGS gene were designed using Integrated DNA Technologies (Coralville, Iowa, USA) guide RNA (gRNA) design tools to minimize off-target, and the potency of these sgRNAs was also tested using the Basic Local Alignment Search Tool (BLAST; U.S. National Center for Biotechnology Information, Bethesda, Md., USA) analysis. The gRNAs were designed to target the conserved region of the FPGS and knockout function of both isoforms. The target sequences and plasmid construct map are shown in
DNA and RNA isolation and analysis: Genomic DNA from putative clones was extracted using DNA-zol Reagent (Thermo Fisher Scientific) and a modified protocol of the previously published method in Ausubel et al. (39). For genotyping, PCR reactions were performed in duplicate with genomic DNA using High-Fidelity Taq DNA polymerase (Thermo Fisher Scientific) according to the manufacturer's protocol. The target-specific primer sets used for PCR are listed in Table 1 and Table 2 below.
The total RNA from putative clones was extracted using the miRNeasy Mini Kit (Qiagen, Germantown, Md., USA). cDNA synthesis from 293T RNA was performed with 1 mg of total RNA using SuperScript III Reverse Transcriptase and the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) according to the manufacturer's protocol.
Cell energy phenotype analysis using the Seahorse XFe96 extracellular flux analyzer. Basal mitochondrial function and metabolic potential of FPGSko-1 and wild-type (WT) cells were measured using the Seahorse Bioscience XFe96 Cell Energy Phenotype Test (Agilent Technologies, Santa Clara, Calif., USA). This assay simultaneously measures the 2 major energy-producing pathways in live cells (mitochondrial respiration and glycolysis), allowing a rapid determination of energy phenotypes of cells and investigating metabolic potential of the cell. The experiments were performed according to the manufacturer's protocol. Briefly, cells were seeded in DMEM supplemented with 10% FBS in 96-well tissue culture plates at a density of 20,000 cells/well and allowed to adhere for 24 hours. Prior to the assay, the medium was changed to DMEM containing 10 mM glucose, 1 mM pyruvate, and 2 mM Gln (pH 7.4), and the cells were equilibrated for 30 minutes at 37° C. The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured under basal conditions. All treatment conditions were analyzed with 6-8 wells/treatment and repeated at least twice. OCR and ECAR values were normalized to cell numbers.
Clariom S Human Array and gene expression analysis: RNA was isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. Total RNA was assessed for the RNA quality verification and microarray hybridization. The Agilent 2100 Bioanalyzer (Agilent Technologies), a microfluidics-based platform, was used for sizing, quantification, and quality of RNA. The RNA integrity number score was generated on the Agilent software. For the microarray analysis, the RNA quality for all of the samples had an RNA integrity number score>7.
For microarray analysis, 3 biologic replicates were included for both control and FPGS mutant. For each array experiment, 500 ng of total RNA was used for labeling using the Clariom S Human Array (Thermo Fisher Scientific). Probe labeling, chip hybridization, and scanning were performed according to the manufacturer's instructions. A Probe Set (gene-exon) was considered expressed if 50% samples had detection above background (DABG) values below the DABG threshold (DABG<0.05).
To validate microarray results, quantitative 2-step RT-PCR was performed. One microgram of total RNA was reverse transcribed to first-strand cDNA with the Qiagen cDNA Synthesis Kit (Qiagen), and this cDNA was subsequently used as a template for quantitative PCR with gene-specific primers. The ubiquitous β-actin gene served as a control for constitutive gene expression. The quantitative RT-PCR (qRT-PCR) reactions were performed using Power Sybr Green PCR Master Mix (Thermo Fisher Scientific) on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, Calif., USA). Relative expression levels (2−ΔCt) were calculated according to the Livak and Schmittgen method (40). Expression levels of each gene were compared with the expression level of actin. Values are the means of 3 biologic and 3 technical replicates and the oligonucleotides used in the study are presented in Tables 1 and 2.
Global DNA methylation measurement: Global 5-methylcytosine (5-mC) levels were quantified using the MethylFlash Methylated DNA Quantification Kits (Epigentek, Farmingdale, N.Y., USA). The DNA concentration was determined using the Qubit Assay (Thermo Fisher Scientific) according to the manufacturer's protocol. Briefly, 100 ng of DNA was used for incubation with both capture and detection antibodies using MethylFlash Methylated DNA Quantification Kit (Colorimetric) from Epigentek. Subsequently, measurements of the absorbance of the sample at 450 nm in a microplate spectrophotometer (BioTek Instruments, Winooski, Vt., USA) were performed with the percentage of the whole genome 5-mC calculation according to manufacturer's instructions. Genomic methylation levels in study samples were expressed as percentage of 5-mC.
Western blotting: Cells were lysed with Mammalian Protein Extraction Reagent lysis buffer (78501; Thermo Fisher Scientific) containing 1 mM PMSF and 1× protease inhibitor cocktail (MilliporeSigma, Burlington, Mass., USA). Proteins (40 mg/well) were separated on 4-12% gradient Bis-Tris NuPage gels (Thermo Fisher Scientific) and blotted on methanol-activated PVDF membrane (Thermo Fisher Scientific) using 1× transfer buffer (LC3625; Thermo Fisher Scientific) according to the manufacturer's instructions. Subsequently, blocking was performed using Li-Cor Biosciences (Lincoln, Nebr., USA) Odyssey blocking buffer (PBS; 927-40000). Thereafter, blocked membranes were incubated with a specific anti-human FPGS antibody (1:1000; AB184564; Abcam, Cambridge, Mass., USA) overnight at 4° C. in the same blocking buffer. The membrane was washed 3 times (5 minutes each wash) with 10 ml PBS with 0.05% Tween and 1× wash with 1×PBS on a shaker and incubated with anti-rabbit antibody dye 680RD (25-68071; Li-Cor Biosciences) for 90 minutes at room temperature. After incubation, the membrane was washed 3 times (5 minutes each wash) with 10 ml PBS with 0.05% Tween and 1× wash with 1×PBS on a shaker. Immunocomplexes were visualized with the Li-Cor Odyssey CLx in 700 channel (red). To visualize b-actin on the membrane, an anti-β-actin monoclonal antibody (012M4821; A1978; MilliporeSigma) and secondary antibody IR-Dye 800 (926-32210; Li-Cor Odyssey; Li-Cor Biosciences) were used.
Immunofluorescence analysis: The relative optimal image-based analysis of 2 key human pluripotent stem cells markers [octamer-binding transcription factor 4 (OCT4) and stage-specific embryonic antigen 4 (SSEA4)] was performed using the Pluripotent Stem Cell Immunocytochemistry Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Briefly, the cells were grown in 10% FBS DMEM supplemented with NEAAs and Glutamax in wells coated with 0.1% gelatin. For immunofluorescence localization, the cells were stained for SSEA4 and Oct4 and counterstained with DAPI using the kit (Thermo Fisher Scientific). The endogenous proteins were labeled using primary antibodies (anti-SSEA4 anti-mouse IgG3 and anti-OCT4 host-rabbit) followed by secondary antibodies conjugated to Alexa Fluor 488 goat anti-mouse IgG3 and Alexa Fluor 594 donkey anti-rabbit.
Chemical complementation assays of FPGS mutants: Cells were maintained as monolayers in DMEM with Gluta-Max (Corning) supplemented with 10% FBS at 37° C. in a 5% CO2 atmosphere. To supplement the growth medium with amino acids, 1× and 2× doses of essential amino acids (Thermo Fisher Scientific) were added in the medium and the cells were grown as previously described for 5 days. In addition to this, Iscove's modified Dulbecco's medium (IMDM) and DMEM with 1×NEAAs (Thermo Fisher Scientific) along with FBS and Glutamax were used as described earlier. For 5-formyl-THF (5-CHO-THF) supplementation experiments, 6S-5-formyl-5,6,7,8-tetrahydrofolic acid (calcium salt; natural calcium folinate) was purchased from Schircks Laboratories (Jona, Switzerland). The stock solution (10 mM) of 5-CHO-THF was made using tissue culture-grade Dulbecco's PBS buffer (Thermo Fisher Scientific), and 100 ml from the stock solution was applied to the 1 ml IMDM to achieve the desired (1 mM) working concentration. The cells were grown for 5 days in the medium supplemented with 5-CHO-THF, and comparative analysis for cell growth was performed with the cells growing with only solvent control (only Dulbecco's PBS without 5-CHO-THF) in DMEM. All the experiments were carried out in triplicate, and cell proliferation was measured using a Cellometer Auto T4 Counting Chamber (Nexcelom Bioscience, Lawrence, Mass., USA).
Genetic complementation of FPGS mutants: To confirm that FPGSk® phenotype was caused by the loss of FPGS function, the WT-FPGS coding region from 293T was cloned and genetic complementation assays were performed on FPGSko-1 and FPGSko-2 mutants. Cells were grown (293T) in DMEM supplemented with 10% FBS, total RNAs were isolated, and first-strand cDNA was reverse transcribed as previously described. The FPGS coding region was amplified using the Phusion High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, Mass., USA) and gene-specific primers (Tables 1 and 2). The resulting amplified product was cloned into pENTR-D-TOPO according to the manufacturer's protocol (Thermo Fisher Scientific), and the fragment was subsequently cloned into the pDest47 vector using gateway cloning according to the manufacturer's instructions (Thermo Fisher Scientific). The resulting expression plasmid (pDest47-FPGS-GFP) containing a functional fusion FPGS [FPGS fused to green fluorescent protein (GFP)] was delivered to FPGSko-1 and FPGSko-2 mutants using Lipofectamine 2000 Transfection reagent (Thermo Fisher Scientific) according to the manufacturer's protocol. Transfected positive clones (transiently expressing FPGS-GFP) were selected 3 days post-transfection using single-cell sorting as we did for selection of the FPGSko mutants. Expression of the transgenic FPGS was confirmed by qRT-PCR.
Metabolomics by hydrophilic interaction liquid chromatography and GC-MS: The cell lines (FPGSko and control) were cultured as described earlier in 100-mm plates. Once they attained confluency, the cells were washed twice with PBS (MilliporeSigma) and detached with 0.25% Trypsin-EDTA at 37° C. for 2 minutes. Subsequently, the cells were collected in 15-ml Falcon tubes, pelleted, and washed 5 times with ice-cold PBS before they were counted in a single-cell suspension using a Cellometer (Nexcelom Bioscience). The experiment was conducted with 5 biologic and 5 technical replicates, and a total of 8 million cells were used in each replicate. Samples were analyzed by the West Coast Metabolomics Center at the University of California-Davis (Davis, CA, USA) for primary metabolites (GC-MS) and biogenic amines [hydrophilic interaction liquid chromatography (HILIC)] using standard operating procedures as described earlier (41, 42).
Glu assay: The relative free Glu concentration in FPGSko mutants was assessed using a Glutamate Assay Kit (Abcam) according to the manufacturer's instructions. Using the kit, the free Glu levels in the mutant were measured and compared with the control cells (293T). The amount of Glu was quantified by colorimetric analysis (spectrophotometry at optical density=450 nm) using a Tecan Microplate Reader (Männedorf, Switzerland).
Cardiac and neural differentiation cell-culture methods: To induce differentiation, FPGSko cells were grown in human basal differentiation medium containing 10% FBS (stem-cell quality; Thermo Fisher Scientific), 1% NEAAs (Thermo Fisher Scientific), 1% penicillin-streptomycin (Thermo Fisher Scientific), 0.05 mM 2-ME (MilliporeSigma), and 2 mM L-GlutaMax in IMDM (Thermo Fisher Scientific) in 6-well ultra-low attachment plates until they reach 40-50% confluency. Subsequently, cells with embryoid body (EB)-like morphology were plated onto 0.1% gelatin-coated 12-well plates and grown in Roswell Park Memorial Institute (RPMI) 1640 medium with GlutaMax (Thermo Fisher Scientific) and serum-free B27 supplement (Thermo Fisher Scientific) differentiation factors and incubated from cardiac lineage to functional cardiomyocytes.
For neural differentiation, FPGSko cells were initially grown onto 0.1% gelatin-coated 12-well plates in DMEM supplemented with 10% FBS (stem-cell quality; Thermo Fisher Scientific), 1% NEAA (Thermo Fisher Scientific), 1% penicillin-streptomycin (Thermo Fisher Scientific), and 1% GlutaMax (Thermo Fisher Scientific). To induce neural differentiation, the cells were dissociated and passaged onto laminin (20 mg/ml; MilliporeSigma)-coated plates and grown in neurobasal medium (Thermo Fisher Scientific) or RPMI 1640 medium (Thermo Fisher Scientific) for 10-15 days at 37° C. and 5% CO2. The medium was supplemented with 1% serum-free B-27 (Thermo Fisher Scientific), 1% Gluta-Max (Thermo Fisher Scientific), 1% NEAA (Thermo Fisher Scientific), and 1% penicillin-streptomycin (Thermo Fisher Scientific).
Cell quantification, imaging, and statistical analysis: Cells were quantified using a Cellometer Auto T4 counting chamber after mixing in a 1:1 ratio with Trypan blue (MilliporeSigma) to exclude dead cells. The average of 2 separate counts was taken to calculate the cell numbers. All live cell imaging was conducted using an EVOS FL Auto microscope (Thermo Fisher Scientific). All statistical analysis was performed using a paired Student's t test with Bonferroni correction or 1-sample Student's t test. Values of P<0.05 were considered significant.
Generation of FPGS depleted)(FPGSko human cells. In mammalian cells, the single gene for FPGS undergoes alternative splicing, resulting in 2 different isoforms, with the mitochondrial and cytosolic isoforms differing only in the N-terminal domain. To investigate the role of FPGS, deletions in both isoforms of FPGS in 293T cells (HEK cells) were generated using CRISPR/Cas9. The gRNAs used targeted conserved exons present in both isoforms. The sequence and targeting strategy are shown in
To confirm that the changes seen were not caused by off-target effects, complementation studies were performed. FPGS cDNA was prepared from WT (293T), cloned into an expression plasmid vector, forming pDest47-FPGS-GFP, which was used for transfection and for functional complementation. The hFPGS expression vector was transformed into FPGSko-1 and FPGSko-2, GFP-positive clones were selected using single-cell sorting, and expression of FPGS was verified using qRT-PCR. All clones complemented with FPGS showed relatively high levels of FPGS expression with cell growth and differentiation similar to that of WT 293T (
FPGSko cells show decreased cell proliferation. HEK cells (293T) exhibit rapid cell growth (43) in 10% FBS DMEM. However, of 15 putative clones for which growth was monitored (unpublished results), all clones exhibited extremely slow growth and a distinctive morphology that was different from the control cells (
FPGSko mutants show reduced metabolic potential. To help understand the extremely slow growth of the mutants, cellular metabolism was examined by monitoring the OCR and ECAR of mutant and WT cells using the SeahorseXFe96. Both the mitochondrial respiration (OCR) and glycolytic activity (ECAR) of the FPGSko-1 cells were significantly decreased in comparison with control cells (
Metabolomic profiling of the FPGSko indicates a decreased methylation capacity and altered amino acid and nucleic acid profiles. HILIC- and GC-MS-based metabolomic approaches were carried out to understand the differential pattern of C1 and other primary metabolites in FPGSko cells as compared with WT cells. A total of 1488 (HILIC) and 503 (GC-MS) compounds were detected in FPGSko and 293T cells, of which 131 biogenic amines (by HILIC) and 165 primary metabolites (by GC-MS) could be assigned chemical structures and quantified based on spectral matching to authentic compounds. Considering the main focus of the study, the analysis was restricted to some key C1 compounds and primary metabolites. Compared with the parental cell, the metabolites belonging to the C1 pathway were changed significantly in FPGSko cells. The ratio of SAM to S-adenosylhomocysteine (SAH) provides an indication of the cellular capacity to catalyze transmethylation reactions. FPGSko cells had a significant accumulation of SAH along with a reduction in SAM. The resulting low SAM/SAH ratio in FPGSko suggests a marked difference in the methylation capacity of the mutant (
The analysis also showed that some nucleotides and amino acids were depleted in FPGSko (
Microarray analysis identified 2315 differential genes in FPGSko cells. A transcriptional analysis of FPGSko was conducted using Clariom S Human Arrays. The cells were grown in DMEM (Corning) supplemented with 10% FBS (stem-cell quality; U.S. origin; Thermo Fisher Scientific) and 1% GlutaMax. These data revealed that 2315 genes were at least 2-fold differentially expressed between the FPGSko mutants and control cells. Among the differentially expressed genes, 1163 had higher expression in the FPGSk® mutant, whereas 1153 had a lower expression (
Close examination of C1 metabolism-related genes showed that around 14 genes were significantly downregulated in the mutant, including thioredoxin-interacting protein (NM_006472), IGF-binding protein 2 (NM_000597), and cystathionine-b-synthase (NM_001178008). Among the genes directly involved in the folate biosynthesis pathway, expression of aldehyde dehydrogenase 1 family, member L2 (ALDH1L2; NM_001034173), MTHFD (NADP+dependent) 2 (NM_006636), and MTHFD (NADP+dependent) 1-like (NM_001242767) were significantly down-regulated in the mutant. Microarray data further validated that the FPGS (NM_001018078) was down-regulated in the FPGSko. In addition to this, expression of 36 genes associated with the methylation process and 26 genes related to DNA repair were affected.
These results were not unexpected based on the direct connection of FPGS to these pathways and process, but some unexpected results were observed in the up-regulated and down-regulated genes. Looking at the top 15 up-regulated genes in the transcript profiling of FPGSko, anosmin-1 (ANOS1) to be 311-fold higher in the mutant, followed by GABA A receptor, β-2 (179-fold), ankyrin repeat domain 1 (ANKRD1; 93-fold), E26 transformation specific (ETS) variant 5 (53-fold), heat shock 22 kDa protein 8 (27-fold), and expression of Dickkopf WNT-signaling pathway inhibitor 1 (DKK1) was 13-fold higher in the mutant as compared with the WT cells (
Most of the genes down-regulated in FPGSko were associated with regulation of cell growth, differentiation, and metabolism. Some genes that manifested notably reduced expression in FPGSko as compared with the WT cells included gametocyte-specific factor 1 (GTSF1; 164-fold), solute carrier family 7 (SLC7; 108-fold), serpin peptidase inhibitor (48-fold), discoidin domain-containing receptor 2 (31-fold), insulin receptor substrate 4 (27-fold), and angiomotin (17-fold). Unbiased clustering analysis of the data suggests that FPGS elimination altered the expression of the genes related to cell differentiation, amino acid transport, angiogenesis, C1 metabolism, neurogenesis, and oxidative stress (
The microarray results for selected genes were validated by real-time quantitative PCR experiments using FPGS mutant and control cells. The transcript levels of GTSF1, SLC7A11, ALDH1L2, and MTHF were significantly repressed in the FPGS mutant, consistent with the microarray results (
Global DNA methylation is reduced and FPGSko cells express pluripotent stem-cell markers. A global decrease in methylated DNA content has previously been observed after treatment with antifolates (15, 18, 19, 47, 48). Therefore, the global level of 5-mC was measured in FPGSko cells and a significant reduction in FPGSko cells was found (
Though slow growing, the morphologic features of FPGSko cells were similar to those of stem cells. Therefore, the markers for stem cells were examined in FPGSko-1 and FPGSko-2 cells grown on 0.1% gelatin-coated plates in DMEM supplemented with 10% FBS, Gln, and NEAAs. The expression of key pluripotency marker genes OCT4 and sex-determining region Y-box 2 (SOX2) were significantly higher in FPGSko clones when compared with parental lines (
FPGSko cells can manifest hallmarks of cardiogenesis and neurogenesis. The 93-fold up-regulation of ANKRD1 [a protein that is highly expressed in stressed cardiac muscle (46)], 13-fold up-regulation of DKK1 [important in regulation of heart development, cardiac repair, and heart disease (49)], and 51-fold down-regulation of thioredoxin-interacting protein [controls cardiac hypertrophy through regulation of thioredoxin activity (50)], was suggestive of ailing cardiac progenitor cells (CPCs). To determine if FPGSko cells could form cardiomyocyte-like cells, an earlier reported cardiac differentiation protocol was adapted (51). Contractile EBs were noted at day 10. The contractile EBs in all groups peaked around day 14 and decreased after 17 days. The expression of cardiac-specific genes was also assessed in the FPGSk® cells by using RT-PCR. Expression of 3 key cardiomyocyte markers [Nkx2 (early cardiac transcriptional factor indicative of cardiac progenitor phenotype), myosin regulatory light chain 2 (a distinctly expressed protein in cardiac muscle), and cardiac troponin T (a muscle contractility regulatory protein indicative of a mature cardiac phenotype)]. All 3 were significantly up-regulated in the FPGSko cells grown in basal 10% FBS and DMEM (
Neurogenesis was seen when FPGSko cells were maintained on laminin-coated plates and grown in neurobasal medium or RPMI 1640 basal medium with Glutamax-I and serum-free B27 supplement differentiation factors (
The neurotransmitters GABA and Glu are known to have a major role in survival, proliferation, and integration of newly formed neurons (52-54), and Glu can act as a positive regulator of neurogenesis (55). Gln also regulates CPC metabolism and proliferation in mammalian systems (56). The gene expression data pertaining to Gln-Glu-GABA metabolism were consistent with this notion, therefore, the free Glu concentration and expression of Glu-ammonia ligase were determined in the mutant. The expression of Glu-ammonia ligase was significantly low in the mutant, and free Glu concentration was significantly high in the FPGSko mutant (
Nutrient supplementation rescues the FPGSko slow-growth phenotype. Whether supplementing the growth medium with various small molecules can rescue the FPGSko slow proliferation phenotype was tested. First, the cell-growth medium was spiked with additional essential amino acids (1×, 2×). Both FPGS mutants supplied with the additional essential amino acids showed increased cell proliferation (
FPGS is a critical enzyme not only because it is required for intracellular folate homeostasis (30) but also because it links to the transmethylation pathway (
Somatic-cell reprogramming into stem cells using 4 transcription factors, Oct4, Sox2, Kruppel-like factor 4, and c-Myc or OCT4, SOX2, NANOG, and LIN28 is well-established (71-73), and suppression of the maintenance DNMT1 or treatment with the DNMT1 inhibitor 5-azacytidine can aid this conversion (74, 75). Folate in its various forms is essential for the conversion of homocysteine to SAM, which is the source of methyl groups for both DNA methylation and histone methylation. Folate deficiency and mutations in folate-dependent pathways are well known to affect mammalian development, even sometimes causing transgenerational effects, probably by affecting epigenetic inheritance (76). As an interesting example, a hypomorphic mutation in the mouse 5-methyl-THF-homocysteine methyltransferase reductase gene, which is required for activation of methionine synthase and thus the formation of SAM, results in congenital malformations that can persist through 5 generations (76). It is clear that a homeostatic balance among C1 metabolism, the methionine cycle, and the transmethylation metabolic pathways are required for normal cell function and development. Elimination of FPGS is expected to affect folate retention and function in both mitochondria and cytoplasm, and this is likely to have profound effects on C1 metabolism. Thus, the metabolism of FPGSko cells is greatly altered. A second finding is that FPGSko cells have features of stem cells.
Energy metabolism of stem cells is predominantly aerobic glycolysis (77). As demonstrated in the working example, the energy metabolism of FPGSko cells is also predominately glycolysis, though at a reduced level (
It is not clear why differentiation is preferentially toward neurons or cardiomyocytes, but 1 possibility is a change in Gln and Glu metabolism. In FPGSko cells, increases in Gln (5-fold), Glu (1.7-fold), and GABA (5-fold) were observed. Glu is the key excitatory and GABA is the main inhibitory neurotransmitter in mammals (80, 81). The transcriptional analysis of FPGSko cells showed that the expression of 10 genes pertaining to Glu metabolism was significantly low. This includes glutathione-specific γ-glutamylcyclotransferase 1 (21-fold), asparagine synthetase (10-fold), and several neuronal and Glu transporters. In addition to this, expression of around 8 genes related to GABA receptors were significantly altered. Because Gln, Glu, and GABA are of special significance for neurons, the expression of neuron-related genes was checked. The expression of 20 genes connected to the brain and neurons were affected. Ras homolog enriched in brain-like 1, which was 15-fold higher in the FPGS mutant, has been associated with the neuronal development and hippocampal neurogenesis (82). In addition to this, 5-fold higher expression of brain acid-soluble protein (83) and 3-fold higher brain-derived neurotrophic factor were observed, which can stimulate neurogenesis in the cell culture (84).
Why do FPGSko-derived pluripotent cells preferentially differentiate into cardiomyocytes? There is a possible involvement of Gln, GABA, and C1 metabolism in CPC proliferation (56, 85-87). The GABA A receptor, which is abundant in the heart and brain, plays a significant role in cardiovascular regulation (88). GABA B receptors are also expressed and functional in mammalian cardiomyocytes (85). Interestingly, Salabei et al. (56) have shown that Gln is a primary regulator of CPC growth, differentiation, and survival.
In FPGSko cells, a distinct immunostaining of OCT4 was observed in both cytosol and the nucleus (
This example demonstrates generating multipotent stem cells (iMS cells) by treating somatic cells transiently with Methotrexate (MTX) and/or pemetrexed (PTX), a demethylating compound that is widely used in clinical practice. MTX and PTX are antifolates that inhibit enzymes (particularly thymidylate synthase and dihydrofolate reductase) involved in folate metabolism and purine and pyrimidine synthesis (100-103). Since these compounds are competitive inhibitors of dihydrofolate synthetase, these compounds were exogenously applied to the WT (HEK 293T and HF57) cells. As shown in
The quiescent nature of the MTX and PTX cells and the association of demethylation with the generation of induced pluripotent stem cells led to examining markers for stem cells formation. The 293T and HF57 cells were grown on 5% dialyzed FBS DMEM medium. DMEM without methionine, DMEM with 15 mg/L (half dose) and DMEM with 30 mg/L (regular DMEM) were used to grow the cells. Once the cells attained 70% confluency, HF57 were treated with 1-μM MTX or PTX and HEK293T were treated with 500 nM MTX or 1 μM PTX. The cells were treated with MTX or PTX for 7-days, and subsequently the medium was replaced with N2B27 medium (Thermo Fisher Scientific, USA). The cells were grown in N2B27 medium for 15 days which supported undifferentiated growth of human embryonic stem cells. Subsequently, these cells were maintained in MTeSR medium supplemented with sodium hypoxanthine and thymidine (HT) for 15 or more days. These conditions supported prolonged self-renewal of putative pluripotent cells, and they were able to form colonies and later embryoid bodies in vitro. Subsequently, the cells were assessed for markers linked to stem cells. Transcriptomics and qRT-PCR were performed to examine whether ES cell marker genes were expressed in these cells. Both MTX and PTX treated cells expressed the marker genes (
A 3-germ layer immunocytochemistry kit (Thermo Fisher Scientific, USA) was used to assess the pluripotency of embryoid bodies derived from MTX and PTX treated cells. A comparative immunochemical analysis of embryoid bodies derived from MTX and PTX treated HF57 and HEK293T cells, human embryonic stem cells (H1), and iPSCs confirmed the presence of all three germ layers (
293T cells were treated with MTX and/or PTX for 10 or more days. SSEA4 positive cells were sorted using single cell sorter and maintained on mTeSR1 medium. After 10-days, cells started forming embryoid bodies indicating altered programing (Epigenetic and metabolic). Right panel shows embryoid bodies of MTX and/or PTX treated cells compared with embryoid bodies derived from human stem cell (
For neural differentiation, MTX/PTX treated, SSEA4 positive cells were initially grown onto Matrigel-coated 6-well plates in mTeSR1 medium. To induce neural differentiation, the cells were dissociated and passaged onto laminin (20 μg ml-1; Sigma-Aldrich, USA) coated plates and grown in Neurobasal medium (Thermo Fisher Scientific, USA) or RPMI1640 medium (Gibco, USA) for 10-15-days at 37° C./5% CO2. The medium was supplemented with 1% serum free B-27 (Gibco, USA), 1% Glutamax (Gibco, USA), 1% NEAA (Gibco, USA), and 1% penicillin/streptomycin (Gibco, USA).
MTX/PTX treated and SSEA4 positive cells were maintained on Matrigel-coated 6-well plates in mTeSR1 medium. To induce differentiation, cells with EB-like morphology were dissociated with Accutase (Invitrogen) at 37° C. for 5 minutes and then single cell suspension was seeded onto a Matrigel-coated cell-culture dish at 100,000 cell/cm2 in mTeSR1 supplemented with 5 μM ROCK inhibitor (Y-27632) for 24 hours. The cells then were cultured in mTeSR1 medium, which was changed daily. Next, 8 μl of 36 mM CHIR99021 were added into 24 ml RPMI/B27-insulin medium to make 12 μM CHIR99021 RPMI/B27-insulin medium and 2-ml of this were added to each well after the old mTeSR1 medium was removed. Exactly 24 hours after adding CHIR, the medium was replaced with 2 ml room temperature RPMI/B27-insulin. The plate was put back into the 37° C., 5% CO2 incubator. 72 hours post addition of CHIR99021, 1 ml medium from each well was removed and the remaining 1-ml was supplemented with 1-ml RPMI/B27-insulin with 5 μM IWP2 (WNT pathway inhibitor). After 48 hours, the medium was replaced with fresh RPMI/B27-insulin and this was incubated at 37° C., 5% CO2 incubator. After this, every 2-days, the old medium was removed from each well of the 12-well plate and 2 ml/well room temperature RPMI/B27 medium was added and incubated at 37° C., 5% CO2. Spontaneous contraction should occur by day 14 and spontaneous beating can be maintained for several weeks. Correlative immunostaining of H1 (positive control), H57-untreated (negative control), H57 (MTX Treated), and 293 (MTX Treated) cell lines clearly show that the treated H57 and 293T cells had cardiac muscle cells in the differentiation medium. The expression of the key cardiomyocyte markers, Nkx2, which is an early cardiac transcriptional factor, indicative of cardiac progenitor phenotype, and cTNT, which is cardiac troponin T, a muscle contractility regulatory protein, indicative of a mature cardiac phenotype, were tested. Both were significantly up-regulated in the MTX or PTX treated cells grown in cardiomyocytes differentiation medium (
This application claims priority to U.S. Provisional Application No. 62/937,940, filed Nov. 20, 2019, which is incorporated by reference herein in its entirety, including drawings.
| Number | Date | Country | |
|---|---|---|---|
| 62937940 | Nov 2019 | US |
