P6826PC00 Enhanced metabolic medium for culturing stem cells and/or progenitor cells Technical field The present invention relates to a medium, also referred to as enhanced metabolic medium, for culturing mammalian pluripotent stem cells and methods of using the same to maintain pluripotency. Background Embryonic stem cells (ESCs) are immortal cell lines derived from the peri-implantation blastocyst and are considered pluripotent as they can contribute to all lineages of the embryo proper. They can be cultured in standard serum-containing media with the cytokine LIF (Serum/LIF) or in various defined conditions, including one that exploits inhibitors of two prominent differentiation-promoting signals alongside LIF (2i/LIF). Using a fluorescent transcriptional reporter for the early endoderm gene Hhex the present inventors previously identified a subpopulation of ESCs in both 2i/LIF culture and media supplemented with knock out serum replacement (KOSR) and LIF (KOSR/LIF), that coexpressed Hhex mRNA with Epi markers, such as NANOG, suggesting this population has earlier unsegregated inner cell mass (ICM)-like qualities. Consistent with this notion, ESCs that co-express Epi and PrE markers can also differentiate into both the embryonic and extra-lineages of the blastocyst, and therefore exhibit greater than pluripotent or totipotent qualities. Although self-renewal in 2i/LIF and KOSR/LIF culture is supported by distinct factors, the present inventors have previously observed genes involved in lipid metabolism to be enriched in ESCs cultured in both of these conditions. Summary The present inventors have found that totipotent ESC subpopulations are characterized by higher rates of oxidative phosphorylation (OXPHOS) and lower rates of glycolysis, compared to standard ESCs. Accordingly, they have found a way to manipulate the metabolic conditions of Serum/LIF media to generate an Enhanced Metabolic Media (EMM), which forces ESCs to upregulate OXPHOS at the expense of glycolysis, leading to increased inner cell mass (ICM)-like transcriptional identity and the generation of cultures comprising enhanced metabolic ESCs (EMESCs). Based on a combination of metabolic, proteomic, and transcriptomic analysis, the present inventors find that EMESCs exploit an OXPHOS cofactor, NAD+, to trigger the activation of a
P6826PC00 lineage specific transcriptional program induced by NAD+-dependent Sirtuin deacetylase activity. In one embodiment, the present disclosure concerns a cell culture medium for maintaining, enhancing and/or promoting pluripotency in a population of mammalian pluripotent stem cells, the medium comprising or consisting of: an aqueous sugar-free basal medium for mammalian cells, D-Galactose at a concentration sufficient for sustaining expansion of the cultured pluripotent stem cells, and a Stat3 activator, wherein said medium is free or substantially free of compounds that promote glycolytic metabolism. In one embodiment, the present disclosure concerns a cell population of pluripotent stem cells and/or progenitor cells, wherein said cells are characterized by: a. proliferating in vitro without further differentiation for at least 3 passages; b. having a cell cycle length of at least 48h after 2 or more passages; c. expressing one or more enhanced pluripotency markers; when cultured in a medium according to any one of the preceding claims. In one embodiment, the present disclosure concerns a cell population of pluripotent stem cells and/or progenitor cells obtained by culturing the cells in a cell culture medium comprising or consisting of: an aqueous sugar-free basal medium for mammalian cells, D-Galactose at a concentration sufficient for sustaining expansion of the cultured pluripotent stem cells, and a Stat3 activator, wherein said medium is free or substantially free of compounds that promote glycolytic metabolism. In one embodiment, the present disclosure concerns an in vitro cell culture comprising: a. mammalian pluripotent stem cells and/or progenitor cells; and b. a cell culture medium comprising or consisting of: an aqueous sugar-free basal medium for mammalian cells, D-Galactose at a concentration sufficient for sustaining expansion of the
P6826PC00 cultured pluripotent stem cells, and a Stat3 activator, wherein said medium is free or substantially free of compounds that promote glycolytic metabolism. In one embodiment, the present disclosure concerns a method method for handling and/or manipulating and/or culturing an embryo for assisted reproduction, a gamete or a stem cell, the method comprising handling and/or manipulating and/or culturing the embryo for IVF or gamete or stem cell in a culture medium comprising or consisting of: an aqueous sugar-free basal medium for mammalian cells, D-Galactose at a concentration sufficient for sustaining expansion of the cultured pluripotent stem cells, and a Stat3 activator, wherein said medium is free or substantially free of compounds that promote glycolytic metabolism. In one embodiment, the present disclosure concerns a method for maintaining, enhancing and/or promoting pluripotency, and/or for enhancing the potency of pluripotent cells in a population of mammalian pluripotent stem cells and/or progenitor cells, the method comprising culturing the pluripotent stem cells and/or progenitor cells in a cell culture medium comprising or consisting of: aqueous sugar-free basal medium for mammalian cells supplemented with D-galactose at a concentration sufficient for sustaining expansion of the cultured pluripotent stem cells, and a Stat3 activator, wherein said medium is free or substantially free of compound that promotes glycolytic metabolism. In one embodiment, the present disclosure concerns a method for rejuvenating mammalian pluripotent stem cells and/or progenitor cells characterized by reduced differentiation capacity, the method comprising culturing the pluripotent stem cells and/or progenitor cells in a cell culture medium comprising or consisting of: aqueous sugar-free basal medium for mammalian cells supplemented with
P6826PC00 D-galactose at a concentration sufficient for sustaining expansion of the cultured pluripotent stem cells, and a Stat3 activator, wherein said medium is free or substantially free of compound that promotes glycolytic metabolism. In one embodiment, the present disclosure concerns a method for promoting enhanced extra-embryonic competence of mammalian pluripotent stem cells and/or progenitor cells, the method comprising culturing the pluripotent stem cells and/or progenitor cells in a cell culture medium comprising or consisting of: aqueous sugar-free basal medium for mammalian cells supplemented with D-galactose at a concentration sufficient for sustaining expansion of the cultured pluripotent stem cells, and a Stat3 activator, wherein said medium is free or substantially free of compound that promotes glycolytic metabolism. In one embodiment, the present disclosure concerns a method of producing a blastocyst-like structure, the method comprising: a) obtaining a cell population of: naïve embryonic stem cells (ESc), naïve induced pluripotent stem cells (iPSs), or extra-embryonic endoderm cells; and b) culturing said cell population in the cell culture medium disclosed herein, thereby producing a blastocyst-like structure characterized by comprising a higher proportion of hypoblast cells compared to a blastocyst-like structure obtained in a glucose-based cell culture medium. In one embodiment, the present disclosure concerns a blastocyst-like structure obtained by the method disclosed herein, wherein said blastocyst-like structure is characterized by comprising a higher proportion of hypoblast cells compared to a blastocyst-like structure obtained in a glucose-based cell culture medium. In one embodiment, the present disclosure concerns a blastocyst-like structure characterized by comprising a higher proportion of hypoblast cells compared to a blastocyst-like structure obtained in a glucose-based cell culture medium.
P6826PC00 Description of Drawings Figure 1 Altering the metabolism of ESCs by forcing a change in the ratio of glycolysis to OXPHOS to create EMESCs increases capacity for self-renewal and differentiation. (a) Basal OCR and ECAR analysis of ESCs during EMM culture at 1 hour, 3 hours, 6 hours and 24 hours, following 3 passages in Serum/LIF. n=24 technical replicates, from 3 independent experiments. (b) Flow cytometry histogram for the Nanog-eGFP reporter ESCs after 1-4d EMM culture, gated on GFP-negative cells. Example profiles taken from n=4 technical replicates, from 2 biologically independent samples. (c) Flow cytometry histogram for the Hhex-Venus reporter ESCs after 1-4d EMM culture, gated on Venus-negative cells. Example profiles taken from n=4 technical replicates, from 2 biologically independent samples. (d) Hhex-Venus reporter ESCs cultured for 2 passages in Serum/LIF or EMM, then immunostained for NANOG and Hhex-Venus (GFP antibody). White asterisks in the EMM/Merge image mark example cells with co-expression. e) Quantification of double positive (DP) cells as a percentage of total Hhex-Venus positive cells, from 6 images of 2 biologically independent samples per condition, **p<0.01, unpaired two-tailed t-test. (f) Immunostaining of Serum/LIF-cultured ESCs or EMESCs (48h) for GATA6, NANOG, OCT4 and SOX2, and g) quantification of 30 individual GATA6+ cells taken from 3 independent experiments. h) Annexin V staining of EMESCs after d1-4 in EMM culture. FACS plot depicts Nanog- eGFP reporter co-expression with Annexin V (representative of 3 independent experiments), and i) quantification of Annexin V staining in Nanoghigh and Nanoglow gates (gated against GFP-negative cells) at all timepoints. (j) ESC and EMESC colonies from individual cells cultured in Serum/LIF or EMM respectively for 9 days, stained for alkaline phosphatase activity (Representation of colonies from each condition). Scale bar = 800µm. k) Quantification of ESC colonies from 3 biologically independent samples per condition, ****p <0.0001, two-tailed chi- square test. (l) Cell cycle profiles of Serum/LIF-cultured ESCs and EMESCs after 2 passages, following staining with Hoechst33342 and analyzed by Flow Cytometry. EMESCs displayed increased proportion in G1 phase (37.21% to 42.73%), and decreased proportion in G2/M (38.10% to 31.72%), *p<0.05, unpaired two-tailed t-test.
P6826PC00 m) Brightfield images of mESC cultured in Serum/LIF, EMM and 2i/LIF, after 10 passages. (n-v) RNAseq analysis of apotosis marker genes Bad, Bak1, Bid, Casoase 3, Caspase 9 and Tnfrsf1a; pluripotency genes Nanog, Nr0b1, Pou5f1, Sal4, Sirt1, and Sox2; and extra-embryonic lineage specific genes Col4a1, Col4a2, Dusp4, Gata6, Lrp2 and Sox7 in ESCs and EMESCs were cultured in Serum/LIF, EMM or 2i/LIF respectively for 10 or 20 passages prior to assays. Data are mean + s.d., n=3 biologically independent samples, *p<0.05, **p<0.01, *** p<0.001 and **** p<0.001 unpaired two-tailed t-test. Figure 2 a) Analysis of confocal optical sections (not shown) of immunostained mouse embryos for CDX2 and NANOG, quantified relative to number of DAPI-positive cells. **p<0.01, n.s.=not significant, unpaired two tailed t-test. E0.5 – E5.5 mouse embryos cultured in KSOM+Glucose/Pyruvate (n=21), KSOM+Galactose (n=29). b) Images of chimeric mouse embryos generated from morula injection with ESCs containing an H2B-Tomato reporter, previously cultured for 2 passages in Serum/LIF (n=27), EMM (n=20) or 2i/LIF (n=34), Embryos were collected at E6.5 and immunostained for GATA6 and KRT7 to mark extra-embryonic endoderm and trophoblast respectively, c) quantification of lineage contribution was based on H2B-Tomato cells in the epiblast, or co-expressed with GATA6 and/or KRT7. ****p <0.0001, two-tailed chi-square test. Figure 3 Scatterplot depicting changes in RNA expression after 24h EMM, with associated ATAC-regions at 24h EMM (Log2FC>1, Padj<0.01). TEs shown as green dots. Figure 4: Metabolic Changes driven by EMM Culture and their Impact on EMESC Phenotypes. a) PC1 vs 2 for Serum/LIF-cultured ESCs at 3h, 9h and 24h in culture. b) PC1 vs 2 for EMM-cultured ESCs at 3h, 9h and 24h in culture. c) Levels of L-Acetylcarnitine in Serum/LIF and EMM-cultured ESCs after 3h culture. ****p<0.001, unpaired two-tailed t-test. d) Levels of L-Carnitine in Serum/LIF-cultured ESCs and EMM-cultured ESCs after 3h culture. ***p<0.005, unpaired two-tailed t-test.
P6826PC00 e) Immunostaining for CPT1A in Serum/LIF and EMM-cultured ESCs after 24h. Data are representative of 3 biological replicates. f) Normalized counts from RNA-seq for Cpt1a in Serum/LIF and EMM-cultured ESCs after 3h, 9h and 24h in the indicated condition. **p<0.01, ***p<0.005, n.s.=not significant, unpaired two-tailed t-test. g) and h) RT-qPCR of EMM-early response genes (Tbx3 and Dnmt3b) in ESCs cultured in Serum/LIF or EMM, +/- the PARP inhibitor Olaparib (10uM) for 24h. Data are the mean expression level, +/- s.d. n=3 biologically independent samples. n.s.=not significant, unpaired two-tailed t-test. i) Western blot analysis of protein levels of H3K9ac, H3K27ac and H4K16ac, relative to total H3 levels in ESCs cultured in Serum/LIF in EMM-/+NAM for 24h. Data are representative of 3 biologically independent samples. j) Flow cytometry histogram for Nanog-eGFP ESCs cultured in Serum/LIF and EMM- /+NAM for 2 passages. Data are representative of 3 experimental replicates. k) Global histone PTM levels quantified by mass spectrometry. n=4 biological replicates (3 for EMM). Two-sided t test. Lines indicate median, boxes represent first and third quartiles and whiskers extend 1.5× IQR. l) RT-qPCR analysis of EMM immediate response genes in ESCs cultured in Serum/LIF and EMM-/+NAM for 24h. Data are mean + s.d., n=3 biologically independent samples. *p<0.05, **p<0.01, ***p<0.005, unpaired two-tailed t-test. m) Immunostaining of EMM+NAM-cultured (48h) ESCs for GATA6, NANOG, OCT4 and SOX2, and n) quantification of 30 individual GATA6+ cells taken from 3 independent experiments. o) RT-qPCR analysis for Nanog mRNA and H3K27ac in Serum/LIF-cultured and EMM cultured ESCs after 1h and 3h culture, +/- A-485 (10nM). Data are mean + s.d., ****p<0.001, unpaired two-tailed t-test. Figure 5: Sirtuin-Dependent Deacetylation of Lineage Specific Transcriptional Regulators Underlies EMM Phenotype. a) Total number of acetylated sites detected in samples. b) Median intensity of all acetylated sites detected in samples. c) PCA plot of acetylated sites in different samples. d) Volcano plots depicting significantly differentially acetylated sites (FDR 5%) between EMM and Serum/LIF and e) between EMM and EMM+NAM (right). See also Supplementary Table 5a.
P6826PC00 f) GO Cellular Component Enrichment table for sites deacetylated in EMM, with percent network. See also Supplementary Table 5A5a, S5Bb. g) Violin plot depicting H3K27ac distribution at Opening, Closing and non-changing enhancers in SL and EMM conditions. ****p<0.0001, one-way ANOVA test. h) Overlap of H3K27ac and H3K4me1 CUT&Tag peaks, defining enhancers in Serum/LIF and EMM. i) to m) Percent of enhancers in each set overlapped by peaks cobound by SOX2, KLF4, and TEAD1. Peaks present in 2/3 replicates per condition for KLF4 and TEAD1, and in 4/6 reps for SOX2. For each condition, the % of enhancers bound to closing enhancer regions are displayed on the left, the % of enhancers bound to opening enhancer regions are represented in the middle, and rest are displayed on the right. n) Violin plot showing SOX2 binding (log2 read count) distribution at all SL-EMM enhancers in WT ESCs. In SL, EMM and EMM+NAM conditions. ****p<0.001, ****p<0.0001, n.s.=not significant, Wilcoxon rank sum test. o) Model depicting SIRT1 regulation of transcription factors and histones to promote an ICM-like identity, downstream of a metabolic shift induced by EMM culture. Figure 6 Expression of embryonic (a-b) and extraembryonic (c) markers in RSet hPSC cultured in N2B27 or EMM. Figure 7 Images of naive hPSC in standard N2B27 or EMM media, at 5% and 20% oxygen concentrations, after 48h culture. Figure 8 Blastoids created from naive hPSC cultured in N2B27 (control) or N2B27-EMM supplemented with PXGL. Blastoid formation in standard N2B27. Figure 9 Lineage specific markers in blastoids created from N2B27 (control) and EMM with and without insulin. a) Blastoids were stained for markers of epiblast (KLF17), hypoblast (GATA4) and trophoblast (GATA3) lineages. b) Proportion of GATA4+ blastoids
P6826PC00 (determined as blastoids containing at least 1 positive cell) in various culture conditions. Detailed description Definitions “Blastoid” or “blastocyst-like cell” as used herein are in-vitro generated, three- dimensional aggregates normally derived by pluripotent stem cells such as embryonic stem cells or induced pluripotent stem cells, which, morphologically and transcriptionally resembles the early, pre-implantation, mammalian conceptus, called the blastocyst. Blastoids are characterized by the presence of key cellular components analogous to a natural blastocyst, including an outer trophectoderm-like layer, an inner cell mass-like structure composed of epiblast and hypoblast, and a cavity similar to the blastocoel. "Assisted reproduction” as used herein refers to medical and technological interventions used to aid or enhance human or animal reproduction. It encompasses all methods and techniques that facilitate conception, implantation, pregnancy, and live birth, either in vivo or in vitro. This includes, but is not limited to, in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), artificial insemination, gamete and embryo cryopreservation, preimplantation genetic diagnosis (PGD), preimplantation genetic screening (PGS), gamete or embryo donation, surrogacy, and other medical or laboratory techniques designed to assist with fertilization, embryo development, or implantation. "Somatic stem cell" as used herein refers to an undifferentiated cell found in a differentiated tissue that can renew itself (clonal) and (with certain limitations) differentiate to yield all the specialized cell types of the tissue from which it originated. "Progenitor cell" as used herein refers to a cell that has the capacity to create progeny that are more differentiated than itself and yet retains a limited capacity to replenish itself. By that definition, stem cells themselves are also progenitor cells, as are the more immediate precursors to terminally differentiated cells. When referring to the cells of the present invention, as described in greater detail below, this broad definition of "progenitor cell" may be used. In a narrower sense, a progenitor cell is often defined as a cell that is intermediate in the differentiation pathway, i.e., it arises from a stem cell
P6826PC00 and is intermediate in the production of a mature cell type or subset of cell types. This type of progenitor cell generally has a limited capacity to self-renew, but in some cases is merely an intermediate in differentiation without this capacity. Accordingly, if this type of cell is referred to herein, it will be referred to as a “progenitor cell” a “non-renewing progenitor cell” or as an “intermediate progenitor” or “precursor cell”. A differentiated cell can be derived from a multipotent cell which itself is derived from a multipotent cell, and so on. While each of these multipotent cells can be considered lineage restricted stem cells, the range of cell types each can give rise to may vary considerably. Some differentiated cells also have the capacity to give rise to cells of greater developmental potential. Such capacity can be natural or can be induced artificially upon treatment with various factors. “Proliferation” indicates an increase in cell number. The term “pluripotent” or “pluripotency” refers to cells with the ability to give rise to progeny that can undergo differentiation, under the appropriate conditions, into cell types that collectively demonstrate characteristics associated with cell lineages from all of the three germ layers (endoderm, mesoderm, and ectoderm), and in most cases, the germ line. When introduced back into an early embryo, pluripotent stem cells can contribute to many or all tissues of a prenatal, postnatal or adult animal. A standard art- accepted test, such as this form of chimera generation or the ability to form a teratoma in adult mice, can be used to establish the pluripotency of a cell population, however identification of various pluripotent stem cell characteristics can also be used to detect pluripotent cells. Pluripotency is found across a continuum cellular states and the stem cell cultures derived from them. These range from naïve pluripotency, that exhibit high efficiencies of clonal growth to cells approaching gastrulation, known as primed. Naïve embryonic and induced pluripotent stem cells (iPSCs) can be made from a range of range of species, although conventional mouse cells are naïve and human primed. All these cell types are pluripotent. On this continuum, naïve and slightly later cell types can generate the full spectrum of lineages including the germ line, whereas later primed cells can form all three germ layers but that may not exhibit all the characteristics of completely pluripotent cells, such as, for example, germline transmission or the ability to generate a whole organism. There are also qualities of stem cell after significant times in culture where a pluripotent stem cell can lose competence or efficiency for differentiation to multiple lineages.
P6826PC00 In some embodiments of the present disclosure, the pluripotency of a cell is enhanced (or increased, or promoted) from an incompletely or partially pluripotent cell to a more pluripotent cell or, in certain embodiments, a completely pluripotent cell, by culturing the cell in the medium of the present disclosure. Pluripotency can be assessed, for example, by teratoma formation, germ-line transmission, and tetraploid embryo complementation. In some embodiments, expression of pluripotency genes or pluripotency markers as discussed elsewhere herein, can be used to assess the pluripotency of a cell. A compound that “promotes glycolytic metabolism" refers to a compound that facilitates cellular metabolic reprogramming from mitochondrial oxidation to glycolysis. In some aspects, a compound that promotes glycolytic metabolism is a compound that promotes glycolysis or a compound that promotes a process upstream of glycolysis (e.g., PDK1 pathway, hypoxia-inducible factor pathway, glucose uptake transporter pathway). In some aspects, a compound that promotes glycolytic metabolism is a compound that inhibits or impedes mitochondrial respiration. In some aspects, a compound that promotes glycolytic metabolism is a compound that promotes a process downstream of glycolysis (e.g., fatty acids synthesis, lipids synthesis, nucleotides synthesis, and amino acids synthesis). Examples of compounds that promote glycolytic metabolism include PDK1 activators, glycolysis activators, glycolysis substrates, glycolytic intermediates and their metabolic precursors thereof, glucose uptake transporter activators, mitochondrial respiration modulators such as oxidative phosphorylation inhibitors, and hypoxia-inducible factor activators. For example, compounds that promote glycolytic metabolism are glucose, pyruvate, insulin, insulin- like growth factor, triiodothyronine, hydrocortisone, neuregulin, and human growth hormone. In some embodiments, the cell culture medium is substantially free of glucose, pyruvate, insulin, insulin-like growth factor, triiodothyronine, hydrocortisone, neuregulin, and human growth hormone. As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to
P6826PC00 capture the potential lack of completeness inherent in many biological and chemical phenomena. As used herein "passage" or "passaging" refers to subculturing of cells. Cells are expanded in culture by being grown in medium. When such cells are subcultured, each round of subculturing is referred to as a passage. The terms "promote" or "increase," or "promoting" or "increasing" are used interchangeably herein. These terms refer to the increase in a measured parameter (e.g., activity, expression, glycolysis, glycolytic metabolism, glucose uptake, biosynthesis downstream of glycolysis) in a treated cell (tissue or subject) in comparison to an untreated cell (tissue or subject). A comparison can also be made of the same cell or tissue or subject between before and after treatment, where the treatment may be culturing in a certain medium rather than another, for example treated cells may be cells cultured in the EMM disclosed herein. The increase is sufficient to be detectable via assays commonly used in the art. In some aspects, the increase in the treated cell is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 3-fold, 4-fold or more in comparison to an untreated cell. As used herein, "inhibit," "prevent" or "reduce," or "inhibiting," "preventing" or "reducing" are used interchangeably herein. These terms refer to the decrease in a measured parameter (e.g., activity, expression, mitochondrial respiration, mitochondrial oxidation, oxidative phosphorylation) in a treated cell (tissue or subject) in comparison to an untreated cell (tissue or subject). A comparison can also be made of the same cell or tissue or subject between before and after treatment, where the treatment may be culturing in a certain medium rather than another, for example treated cells may be cells cultured in the EMM disclosed herein. The decrease is sufficient to be detectable. In some aspects, the decrease in the treated cell is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or completely inhibited in comparison to an untreated cell. In some aspects the measured parameter is undetectable (i.e., completely inhibited) in the treated cell in comparison to the untreated cell. Cell culture medium The present invention takes advantage of forcing a metabolic change in embryonic stem cells to promote a developmental identity that approximates the inner cell mass
P6826PC00 (ICM) of the early mammalian blastocyst in cultures, referred to as enhanced metabolic ESCs (EMESCs). Creation of EMESCs depends on inhibition of glycolysis and stimulation of oxidative phosphorylation (OXPHOS), that in turn activates NAD+- dependent deacetylases of the Sirtuin family. This is accomplished by utilization of Enhanced Metabolic Media (EMM) described herein, which stimulates an enhanced ICM-like metabolic signature in which cells elevate their levels of OXPHOS and reduce dependence on glycolysis by forcing extensive utilization of OXPHOS as a means to generate sufficient ATP for cell survival. In one embodiment, the present disclose concerns a cell culture medium for maintaining, enhancing and/or promoting pluripotency in a population of mammalian pluripotent stem cells, the medium comprising or consisting of: an aqueous sugar-free basal medium for mammalian cells, D-Galactose at a concentration sufficient for sustaining expansion of the cultured pluripotent stem cells, and a Stat3 activator, wherein said medium is free or substantially free of compounds that promote glycolytic metabolism. Media for culturing ESCs commonly used in the art comprise D-glucose and pyruvate, however the present inventors have found that replacing D-glucose and pyruvate with D-galactose reduce dependence on glycolysis. Without being bound by theory, D- galactose can only enter the glycolytic pathway as a result of its conversion to glucose- 6-phosphate, a process requiring 2 molecules of ATP. Given that glycolysis itself only produces two molecules of ATP, glycolysis will produce no net gain of ATP and is therefore strongly inhibited. In some embodiments, the cell culture medium is substantially free of glucose. In some embodiments, the cell culture medium is substantially free of pyruvate. In some embodiments, the compounds that promote glycolytic metabolism are glucose, pyruvate, insulin, insulin-like growth factor, triiodothyronine, hydrocortisone, neuregulin, and human growth hormone. In some embodiments, the cell culture medium is substantially free of glucose, pyruvate, insulin, insulin-like growth factor, triiodothyronine, hydrocortisone, neuregulin, and human growth hormone. D-Galactose is a sugar analogue of glucose, which acts as a competitive inhibitor of
P6826PC00 hexokinase, the first enzyme in the glycolytic pathway. The glycolytic pathway is used by the cells in a variety of ways, from the generation of 2 molecules of pyruvate from 1 molecule of glucose, which generates 2 molecules of ATP, to the generation of macromolecules via connected pathways such as the pentose phosphate pathway. In some embodiments, the medium consists of: an aqueous sugar-free basal medium for mammalian cells, D-Galactose at a concentration sufficient for sustaining expansion of the cultured pluripotent stem cells, and a Stat3 activator. In some embodiments, the aqueous sugar-free basal medium is supplemented with D- galactose at a concentration of 1 to 25 g/L, such as 1 to 20 g/L, such as 1 to 15 g/L, such as 1 to 10 g/L, such as 1 to 7 g/L, such as 2 to 25 g/L, such as 3 to 25 g/L, such as 4 to 25 g/L, such as 3 to 20 g/L, such as 3 to 15 g/L, such as 3 to 10 g/L, such as 3 to 7 g/L, such as of about 4.5 g/L. In some embodiments, the aqueous sugar-free basal medium is supplemented with D- galactose at a concentration of between 1.75 and 4.5 g/L, such as between 1.75 and 3.5 g/L, between 1.75 and 3.25 g/L, between 1.75 and 2.75 g/L, between 1.75 and 2.25 g/L, between 2.25 and 4.5 g/L, between 2.25 and 3.5 g/L, between 2.25 and 3.25 g/L, between 2.25 and 2.75 g/L, between 2.75 and 4.5 g/L, between 2.75 and 3.5 g/L, between 2.75 and 3.25 g/L, between 3 and 4.5 g/L, between 3 and 4 g/L, between 3 and 3.5 g/L, between 3 and 3.25 g/L, between 3 and 2.75 g/L, between 3 and 2.25 g/L, between 3 and 1.75 g/L, between 3.25 and 4.5 g/L, between 3.25 and 3.5 g/L, between 3.5 and 4.5 g/L, between 4 and 4.5 g/L, between 4 and 3.5 g/L, between 4 and 3.25 g/L, between 4 and 2.75 g/L, between 4 and 2.25 g/L, between 4 and 1.75 g/L, or between 4.25 and 4.5 g/L. Signal transducer and activator of transcription (STAT) proteins are a class of transcription factor that are activated by cytokines, growth factors and other peptide ligands. Without being bound by theory, STAT3 is essential in maintaining self-renewal of embryonic stem cells (ESCs) and modulates ESC differentiation. In some embodiments, the Stat3 activator is interleukin 6 (IL6), interleukin 7 (IL7),
P6826PC00 interleukin 9 (IL9), interleukin 10 (IL10), interleukin 11 (IL11), interleukin 15 (IL15), interleukin 22 (IL22), IFN-α/β, Leukemia inhibitory factor (LIF), hepatocyte growth factor (HGF), oncostatin M (OSM), leptin, and/or growth hormone (GH), and combinations thereof. In some embodiments, the Stat3 activator stimulates the activation of Stat3 targets via JAK/STAT signaling. In some embodiments, the Stat3 activator is interleukin 6 (IL6), interleukin 7 (IL7), interleukin 9 (IL9), interleukin 10 (IL10), interleukin 11 (IL11), interleukin 15 (IL15), interleukin 22 (IL22), IFN-α/β, Leukemia inhibitory factor (LIF), hepatocyte growth factor (HGF), oncostatin M (OSM), leptin, and/or growth hormone (GH), and combinations thereof, wherein the Stat3 activator stimulates the activation of Stat3 targets via JAK/STAT signaling. In some embodiments, the medium further comprises L-carnitine. In some embodiments, the medium further comprises a lipid supplement. In some embodiments, the lipid supplement comprises one or more of oleic acid, palmitic acid, alpha-linoleic acid, arachidonic acid, arachidic acid, palmitoleic acid, myristic acid, myristoleic acid, linoleic acid, stearic acid, cholesterol, DL-alpha-tocopheryl and Kolliphor P188. In some embodiments, the lipid supplement comprises one or more of oleic acid, palmitic acid, alpha-linoleic acid, and arachidonic acid and a carrier. In some embodiments, the carrier is an albumin, a liposome, an extracellular vesicle, an exosome, a nanostructured lipid carrier, or a cyclodextrin. The presence of L-carnitine and/or a lipid supplement in the cell culture medium of the present disclosure may stimulate Oxphos via lipid metabolism, including via the promotion of transport for fatty acids into the mitochondria by Carnitine Palmitoyltransferase I (CPT1), where they will be metabolized in the Tri Carboxylic Acid (TCA) cycle. The basal medium as used herein is an aqueous sugar-free basal medium and it may provide standard inorganic salts such as zinc, iron, magnesium, calcium, and potassium, vitamins, glucose, buffer system, and key amino acids. Furthermore, the basal medium may comprise fetal bovine serum, serum replacement or a functional variant thereof. The basal medium may be serum free. In some embodiments, the basal medium comprises fetal bovine serum, serum replacement or a functional variant thereof. In some embodiments, the basal medium is serum-free. In some
P6826PC00 embodiments, the aqueous sugar-free basal medium comprises advanced DMEM, Biogro™, SkGM™, Ham's F10, Ham's F12, Iscove's modified Dulbecco's medium, neurobasal medium, RPMI 1640, MCDB120 medium, or N2B27 without pyruvate, without glucose. Examples of the serum replacement include those appropriately containing, for example, albumin, transferrin, fatty acid, collagen precursor, trace element, 2- mercaptoethanol or 3' thiolglycerol, an equivalent thereof and so on. Such serum replacement can be prepared by, for example, the method described in WO98/30679. In addition, the serum replacement can be a commercially available product. Examples of such commercially available serum replacement include Knockout™ Serum Replacement (manufactured by Invitrogen: hereinafter sometimes referred to as KSR), Chemically defined lipid concentrate (manufactured by Gibco), and Glutamine (manufactured by Gibco). In some embodiments, the aqueous sugar-free basal medium comprises or consists of: a. glucose-depleted Dulbecco's Modified Eagle Medium (DMEM); b. Fetal Bovine Serum (FBS), c. 2-mercaptoethanol, d. Minimum Essential Medium (MEM) nonessential amino acids, e. L-glutamine, and f. water. In some embodiments, the aqueous sugar-free basal medium comprises about 10% (v/v) FBS. In some embodiments, the aqueous sugar-free basal medium comprises about 100 µM 2-mercaptoethanol. In some embodiments, the aqueous sugar-free basal medium comprises about 100 mM Minimum Essential Medium (MEM) nonessential amino acids. In some embodiments, the aqueous sugar-free basal medium comprises about 2 mM L-glutamine. In some embodiments, the aqueous sugar-free basal medium comprises between 5% and 15% FBS, such as between 5% and 14%, between 5% and 13%, between 5% and 12%, between 5% and 11%, between 5% and 10%, between 5% and 9%, between 5% and 8%, between 5% and 7%, between 5% and 6%, between 6% and 15%, between 6% and 14%, between 6% and 13%, between 6% and 12%, between 6% and 11%, between 6% and 10%, between 6% and 9%, between 6% and 8%, between 6% and 7%, between 7% and 15%, between 7% and 14%, between 7% and 13%, between 7% and 12%, between
P6826PC00 7% and 11%, between 7% and 10%, between 7% and 9%, between 7% and 8%, between 8% and 15%, between 8% and 14%, between 8% and 13%, between 8% and 12%, between 8% and 11%, between 8% and 10%, between 8% and 9%, between 9% and 15%, between 9% and 14%, between 9% and 13%, between 9% and 12%, between 9% and 11%, between 9% and 10%, between 10% and 15%, between 10% and 14%, between 10% and 13%, between 10% and 12%, between 10% and 11%, between 11% and 15%, between 11% and 14%, between 11% and 13%, between 11% and 12%, between 12% and 15%, between 12% and 14%, between 12% and 13%, between 13% and 15%, between 13% and 14%, between 14% and 15% FBS. In some embodiments, the aqueous sugar-free basal medium comprises 10% (v/v) FBS. In some embodiments, the basal medium comprises between 50 and 150 µM 2- mercaptoethanol, such as between 50 and 140 µM, between 50 and 130 µM, between 50 and 120 µM, between 50 and 110 µM, between 50 and 100 µM, between 50 and 90 µM, between 50 and 80 µM, between 50 and 70 µM, between 50 and 60 µM, between 60 and 150 µM, between 60 and 140 µM, between 60 and 130 µM, between 60 and 120 µM, between 60 and 110 µM, between 60 and 100 µM, between 60 and 90 µM, between 60 and 80 µM, between 60 and 70 µM, between 70 and 150 µM, between 70 and 140 µM, between 70 and 130 µM, between 70 and 120 µM, between 70 and 110 µM, between 70 and 100 µM, between 70 and 90 µM, between 70 and 80 µM, between 80 and 150 µM, between 80 and 140 µM, between 80 and 130 µM, between 80 and 120 µM, between 80 and 110 µM, between 80 and 100 µM, between 80 and 90 µM, between 90 and 150 µM, between 90 and 140 µM, between 90 and 130 µM, between 90 and 120 µM, between 90 and 110 µM, between 90 and 100 µM, between 100 and 150 µM, between 100 and 140 µM, between 100 and 130 µM, between 100 and 120 µM, between 100 and 110 µM, between 110 and 150 µM, between 110 and 140 µM, between 110 and 130 µM, between 110 and 120 µM, between 120 and 150 µM, between 120 and 140 µM, between 120 and 130 µM, between 130 and 150 µM, between 130 and 140 µM, between 140 and 150 µM 2-mercaptoethanol. In some embodiments, the aqueous sugar-free basal medium comprises 100 µM 2- mercaptoethanol. In some embodiments, the aqueous sugar-free basal medium comprises between 50 and 150 mM Minimum Essential Medium (MEM) nonessential amino acids, such as between 50 and 140 mM, between 50 and 130 mM, between 50 and 120 mM, between 50 and 110 mM, between 50 and 100 mM, between 50 and 90 mM, between 50 and 80 mM, between 50 and 70 mM, between 50 and 60 mM, between 60 and 150 mM, between 60 and 140 mM, between 60 and 130 mM, between
P6826PC00 60 and 120 mM, between 60 and 110 mM, between 60 and 100 mM, between 60 and 90 mM, between 60 and 80 mM, between 60 and 70 mM, between 70 and 150 mM, between 70 and 140 mM, between 70 and 130 mM, between 70 and 120 mM, between 70 and 110 mM, between 70 and 100 mM, between 70 and 90 mM, between 70 and 80 mM, between 80 and 150 mM, between 80 and 140 mM, between 80 and 130 mM, between 80 and 120 mM, between 80 and 110 mM, between 80 and 100 mM, between 80 and 90 mM, between 90 and 150 mM, between 90 and 140 mM, between 90 and 130 mM, between 90 and 120 mM, between 90 and 110 mM, between 90 and 100 mM, between 100 and 150 mM, between 100 and 140 mM, between 100 and 130 mM, between 100 and 120 mM, between 100 and 110 mM, between 110 and 150 mM, between 110 and 140 mM, between 110 and 130 mM, between 110 and 120 mM, between 120 and 150 mM, between 120 and 140 mM, between 120 and 130 mM, between 130 and 150 mM, between 130 and 140 mM, between 140 and 150 mM Minimum Essential Medium (MEM) nonessential amino acids. In some embodiments, the aqueous sugar-free basal medium comprises 100 mM Minimum Essential Medium (MEM) nonessential amino acids. In some embodiments, the aqueous sugar-free basal medium comprises between 0.5 and 10 mM L-glutamine, such as between 0.5 and 9 mM, between 0.5 and 8 mM, between 0.5 and 7 mM, between 0.5 and 6 mM, between 0.5 and 5 mM, between 0.5 and 4 mM, between 0.5 and 3 mM, between 0.5 and 2 mM, between 0.5 and 1 mM, between 1 and 10 mM, between 1 and 9 mM, between 1 and 8 mM, between 1 and 7 mM, between 1 and 6 mM, between 1 and 5 mM, between 1 and 4 mM, between 1 and 3 mM, between 1 and 2 mM, between 2 and 10 mM, between 2 and 9 mM, between 2 and 8 mM, between 2 and 7 mM, between 2 and 6 mM, between 2 and 5 mM, between 2 and 4 mM, between 2 and 3 mM, between 3 and 10 mM, between 3 and 9 mM, between 3 and 8 mM, between 3 and 7 mM, between 3 and 6 mM, between 3 and 5 mM, between 3 and 4 mM, between 4 and 10 mM, between 4 and 9 mM, between 4 and 8 mM, between 4 and 7 mM, between 4 and 6 mM, between 4 and 5 mM, between 5 and 10 mM, between 5 and 9 mM, between 5 and 8 mM, between 5 and 7 mM, between 5 and 6 mM, between 6 and 10 mM, between 6 and 9 mM, between 6 and 8 mM, between 6 and 7 mM, between 7 and 10 mM, between 7 and 9 mM, between 7 and 8 mM, between 8 and 10 mM, between 8 and 9 mM, between 9 and 10 mM L-glutamine. In some embodiments, the aqueous sugar-free basal medium comprises 2 mM L-glutamine. In some embodiments, the aqueous sugar-free basal medium comprises or consists of:
P6826PC00 a. glucose-depleted Dulbecco's Modified Eagle Medium (DMEM); b. 10% Fetal Bovine Serum (FBS), c. 100 µM 2-mercaptoethanol, d. 100 mM Minimum Essential Medium (MEM) nonessential amino acids, e. 2 mM L-glutamine, f. water. In some embodiments, the aqueous sugar-free serum-free basal medium comprises or consists of: a. glucose-depleted Dulbecco's Modified Eagle Medium/Nutrient Mixture F- 12 (DMEM/F12), b. glucose and pyruvate depleted Neurobasal-A media, c. insulin depleted B27 supplement, d. N2 media, e. L-glutamine, and f. 2-mercaptoethanol. In some embodiments, the aqueous sugar-free serum-free basal medium comprises or consists of about 100 mL DMEM-F12. In some embodiments, the aqueous sugar-free serum-free basal medium comprises or consists of about 100 mL glucose and pyruvate depleted Neurobasal-A media. In some embodiments, the aqueous sugar-free serum- free basal medium comprises or consists of about 1x N2 media. In some embodiments, the aqueous sugar-free serum-free basal medium comprises or consists of about 1x insulin depleted B27 supplement. In some embodiments, the aqueous sugar-free serum-free basal medium comprises or consists of about 1x L-glutamine. In some embodiments, the aqueous sugar-free serum-free basal medium comprises or consists of about 100 µM 2-mercaptoethanol. In some embodiments, the aqueous sugar-free serum-free basal medium comprises or consists of between 0.2 and 2.0x N2 media, such as between 0.2 and 1.8x, between 0.2 and 1.6x, between 0.2 and 1.4x, between 0.2 and 1.2x, between 0.2 and 1.0x, between 0.2 and 0.8x, between 0.2 and 0.6x, between 0.2 and 0.4x, between 0.4 and 2.0x, between 0.4 and 1.8x, between 0.4 and 1.6x, between 0.4 and 1.4x, between 0.4 and 1.2x, between 0.4 and 1.0x, between 0.4 and 0.8x, between 0.4 and 0.6x, between 0.6 and 2.0x, between 0.6 and 1.8x, between 0.6 and 1.6x, between 0.6 and 1.4x, between 0.6 and 1.2x, between 0.6 and 1.0x, between 0.6 and 0.8x, between 0.8 and 2.0x, between 0.8 and 1.8x, between 0.8 and
P6826PC00 1.6x, between 0.8 and 1.4x, between 0.8 and 1.2x, between 0.8 and 1.0x, between 1.0 and 2.0x, between 1.0 and 1.8x, between 1.0 and 1.6x, between 1.0 and 1.4x, between 1.0 and 1.2x, between 1.2 and 2.0x, between 1.2 and 1.8x, between 1.2 and 1.6x, between 1.2 and 1.4x, between 1.4 and 2.0x, between 1.4 and 1.8x, between 1.4 and 1.6x, between 1.6 and 2.0x, between 1.6 and 1.8x, or between 1.8 and 2.0x N2 media. In some embodiments, the aqueous sugar-free serum-free basal medium comprises or consists of between 0.2 and 2.0x insulin depleted B27 supplement, such as between 0.2 and 1.8x, between 0.2 and 1.6x, between 0.2 and 1.4x, between 0.2 and 1.2x, between 0.2 and 1.0x, between 0.2 and 0.8x, between 0.2 and 0.6x, between 0.2 and 0.4x, between 0.4 and 2.0x, between 0.4 and 1.8x, between 0.4 and 1.6x, between 0.4 and 1.4x, between 0.4 and 1.2x, between 0.4 and 1.0x, between 0.4 and 0.8x, between 0.4 and 0.6x, between 0.6 and 2.0x, between 0.6 and 1.8x, between 0.6 and 1.6x, between 0.6 and 1.4x, between 0.6 and 1.2x, between 0.6 and 1.0x, between 0.6 and 0.8x, between 0.8 and 2.0x, between 0.8 and 1.8x, between 0.8 and 1.6x, between 0.8 and 1.4x, between 0.8 and 1.2x, between 0.8 and 1.0x, between 1.0 and 2.0x, between 1.0 and 1.8x, between 1.0 and 1.6x, between 1.0 and 1.4x, between 1.0 and 1.2x, between 1.2 and 2.0x, between 1.2 and 1.8x, between 1.2 and 1.6x, between 1.2 and 1.4x, between 1.4 and 2.0x, between 1.4 and 1.8x, between 1.4 and 1.6x, between 1.6 and 2.0x, between 1.6 and 1.8x, or between 1.8 and 2.0x insulin depleted B27 supplement. In some embodiments, the aqueous sugar-free serum-free basal medium comprises or consists of between 0.2 and 2.0x L-glutamine, such as between 0.2 and 1.8x, between 0.2 and 1.6x, between 0.2 and 1.4x, between 0.2 and 1.2x, between 0.2 and 1.0x, between 0.2 and 0.8x, between 0.2 and 0.6x, between 0.2 and 0.4x, between 0.4 and 2.0x, between 0.4 and 1.8x, between 0.4 and 1.6x, between 0.4 and 1.4x, between 0.4 and 1.2x, between 0.4 and 1.0x, between 0.4 and 0.8x, between 0.4 and 0.6x, between 0.6 and 2.0x, between 0.6 and 1.8x, between 0.6 and 1.6x, between 0.6 and 1.4x, between 0.6 and 1.2x, between 0.6 and 1.0x, between 0.6 and 0.8x, between 0.8 and 2.0x, between 0.8 and 1.8x, between 0.8 and 1.6x, between 0.8 and 1.4x, between 0.8 and 1.2x, between 0.8 and 1.0x, between 1.0 and 2.0x, between 1.0 and 1.8x, between 1.0 and 1.6x, between 1.0 and 1.4x, between 1.0 and 1.2x, between 1.2 and 2.0x, between 1.2 and 1.8x, between 1.2 and 1.6x, between 1.2 and 1.4x, between 1.4 and 2.0x, between 1.4 and 1.8x, between 1.4 and 1.6x, between 1.6 and 2.0x, between 1.6 and 1.8x, or between 1.8 and 2.0x L-glutamine. In some embodiments, the aqueous sugar-free serum-free basal medium comprises or consists of between 25 and 250 mL DMEM/F12, such as between 25 and 225 mL, between 25 and 200 mL,
P6826PC00 between 25 and 175 mL, between 25 and 150 mL, between 25 and 125 mL, between 25 and 100 mL, between 25 and 75 mL, between 25 and 50 mL, between 50 and 250 mL, between 50 and 225 mL, between 50 and 200 mL, between 50 and 175 mL, between 50 and 150 mL, between 50 and 125 mL, between 50 and 100 mL, between 50 and 75 mL, between 75 and 250 mL, between 75 and 225 mL, between 75 and 200 mL, between 75 and 175 mL, between 75 and 150 mL, between 75 and 125 mL, between 75 and 100 mL, between 100 and 250 mL, between 100 and 225 mL, between 100 and 200 mL, between 100 and 175 mL, between 100 and 150 mL, between 100 and 125 mL, between 125 and 250 mL, between 125 and 225 mL, between 125 and 200 mL, between 125 and 175 mL, between 125 and 150 mL, between 150 and 250 mL, between 150 and 225 mL, between 150 and 200 mL, between 150 and 175 mL, between 175 and 250 mL, between 175 and 225 mL, between 175 and 200 mL, between 200 and 250 mL, between 200 and 225 mL, or between 225 and 250 mL DMEM/F12. In some embodiments, the aqueous sugar-free serum-free basal medium comprises or consists of between 25 and 250 mL glucose and pyruvate depleted Neurobasal-A media, such as between 25 and 225 mL, between 25 and 200 mL, between 25 and 175 mL, between 25 and 150 mL, between 25 and 125 mL, between 25 and 100 mL, between 25 and 75 mL, between 25 and 50 mL, between 50 and 250 mL, between 50 and 225 mL, between 50 and 200 mL, between 50 and 175 mL, between 50 and 150 mL, between 50 and 125 mL, between 50 and 100 mL, between 50 and 75 mL, between 75 and 250 mL, between 75 and 225 mL, between 75 and 200 mL, between 75 and 175 mL, between 75 and 150 mL, between 75 and 125 mL, between 75 and 100 mL, between 100 and 250 mL, between 100 and 225 mL, between 100 and 200 mL, between 100 and 175 mL, between 100 and 150 mL, between 100 and 125 mL, between 125 and 250 mL, between 125 and 225 mL, between 125 and 200 mL, between 125 and 175 mL, between 125 and 150 mL, between 150 and 250 mL, between 150 and 225 mL, between 150 and 200 mL, between 150 and 175 mL, between 175 and 250 mL, between 175 and 225 mL, between 175 and 200 mL, between 200 and 250 mL, between 200 and 225 mL, or between 225 and 250 mL glucose and pyruvate depleted Neurobasal-A media. In some embodiments, the aqueous sugar-free serum-free basal medium comprises or consists of between 25 and 250 µM 2-mercaptoethanol, such as between 25 and 225 µM, between 25 and 200 µM, between 25 and 175 µM, between 25 and 150 µM, between 25 and 125 µM, between 25 and 100 µM, between 25 and 75 µM, between 25 and 50 µM, between 50 and 250 µM, between 50 and 225 µM, between 50 and 200
P6826PC00 µM, between 50 and 175 µM, between 50 and 150 µM, between 50 and 125 µM, between 50 and 100 µM, between 50 and 75 µM, between 75 and 250 µM, between 75 and 225 µM, between 75 and 200 µM, between 75 and 175 µM, between 75 and 150 µM, between 75 and 125 µM, between 75 and 100 µM, between 100 and 250 µM, between 100 and 225 µM, between 100 and 200 µM, between 100 and 175 µM, between 100 and 150 µM, between 100 and 125 µM, between 125 and 250 µM, between 125 and 225 µM, between 125 and 200 µM, between 125 and 175 µM, between 125 and 150 µM, between 150 and 250 µM, between 150 and 225 µM, between 150 and 200 µM, between 150 and 175 µM, between 175 and 250 µM, between 175 and 225 µM, between 175 and 200 µM, between 200 and 250 µM, between 200 and 225 µM, or between 225 and 250 µM 2-mercaptoethanol. In some embodiments, the aqueous sugar-free serum-free basal medium comprises or consists of 100 mL DMEM-F12. In some embodiments, the aqueous sugar-free serum-free basal medium comprises or consists of 100 mL glucose and pyruvate depleted Neurobasal-A media. In some embodiments, the aqueous sugar-free serum-free basal medium comprises or consists of 1x N2 media. In some embodiments, the aqueous sugar-free serum-free basal medium comprises or consists of 1x insulin depleted B27 supplement. In some embodiments, the aqueous sugar-free serum-free basal medium comprises or consists of 1x L-glutamine. In some embodiments, the aqueous sugar- free serum-free basal medium comprises or consists of 100 µM 2-mercaptoethanol. In some embodiments, the aqueous sugar-free serum-free basal medium comprises or consists of: a. 100 mL glucose-depleted Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12), b. 100 mL glucose and pyruvate depleted Neurobasal-A media, c. 1x insulin depleted B27 supplement, d. 1x N2 media, e. 1x L-glutamine, and f. 100 µM 2-mercaptoethanol. Cell population The medium of the present invention may be used for culturing cell populations such as pluripotent stem cells and/or progenitor cells. A pluripotent stem cell may be a mammalian pluripotent stem cell, for example a human pluripotent stem cell. In some
P6826PC00 embodiments, the pluripotent stem cell is a mammalian stem cell line known in the art. In some embodiments, the pluripotent stem cell is an induced pluripotent stem (iPS) cell, or a stably reprogrammed cell which is an intermediate pluripotent stem cell and can be further reprogrammed into an iPS cell, e.g., partial induced pluripotent stem cells (also referred to as "piPS cells"). In some embodiments, the pluripotent stem cell, iPSC or piPSC is a genetically modified pluripotent stem cell. A progenitor cell is a cell that has the capacity to create progeny that are more differentiated than itself and yet retains the capacity to replenish the pool of progenitors. In a narrower sense, a progenitor cell is often defined as a cell that is intermediate in the differentiation pathway, i.e., it arises from a stem cell and is intermediate in the production of a mature cell type or subset of cell types. In one embodiment, the present disclosure concerns a cell population comprising or consisting essentially of pluripotent stem cells and/or progenitor cells, wherein said cells are characterized by: a. proliferating in vitro without further differentiation for at least 3 passages; b. having a cell cycle length of at least 48h after 2 or more passages; c. expressing one or more enhanced pluripotency markers; when cultured in the EMM of the present disclosure, such as in a medium comprising or consisting of: an aqueous sugar-free basal medium for mammalian cells, D-Galactose at a concentration sufficient for sustaining expansion of the cultured pluripotent stem cells, and a Stat3 activator, wherein said medium is free or substantially free of compounds that promote glycolytic metabolism. The present inventors have found that a cell population comprising or consisting essentially of pluripotent stem cells and/or progenitor cells according to the present disclosure and obtained by culturing pluripotent stem cells and/or progenitor cells in a cell culture medium according to the present disclosure is characterized by having a reduced transcriptional noise and at the same time elevated cell type specific transcriptional signatures, compared to pluripotent stem cells and/or progenitor cells cultured in glucose-based cell culture media. Thus, a cell population according to the present disclosure may exhibit reduced aging.
P6826PC00 In one embodiment, the present disclosure concerns a cell population comprising or consisting essentially of pluripotent stem cells and/or progenitor cells, wherein said cells are characterized by an enhanced dependence on OXPHOS and a reduced dependence on glycolysis. In one embodiment, the present disclosure concerns a cell population comprising or consisting essentially of pluripotent stem cells and/or progenitor cells, wherein said cells are characterized by a diapause-phenotype. The present inventors have found that an enhanced dependence on OXPHOS characterized by higher levels of fatty acid oxidation and the suppression of glycolysis induces an ICM- like foundation state for lineage specification. This implies that reducing metabolic or transcriptional noise at the same time as stimulating specific transcriptional signals (enhanced signal:noise ratio) enhances differentiation competence and that this metabolically “quiet” state (i.e. a diapause-like phenotype), was selected to have the greatest developmental potential. Moreover, the decay of this transcriptional signal to noise ratio could be an essential component of aging, as progenitor or stem cells produce a spectrum of unwanted phenotypes in response to reducing this ratio. The pluripotent stem cells and/or progenitor cells of the present invention may have characteristic expression patterns of one or more RNAs. These RNAs may for example be associated with extra-embryonic differentiation. In some embodiments, said pluripotent stem cells and/or progenitor cells are further characterized by expressing one or more RNAs associated with extra-embryonic differentiation, such as with the hypoblast or trophoblast, for example one or more of Hhex, Dab2, Gata4, Gata6 and Pdgfra (also referred to herein as PDGFRA). In some embodiments, said cells are further characterized by expressing one or more RNAs associated with extra- embryonic differentiation, for example one or more of Gata6, Gata4, Sox7, Emp1, Col4a1, Sox17, Pdgfra, Tbx15, Foxq1, Marcks, Krt8, Krt18, Emoes, Dab2, Dusp4, Gata3, Tead4, Lrp2, Tagin2, Bmyc, Cldn2, and Tbx1. See for example the data reported in Examples 6 and 7 herein.
P6826PC00 In one embodiment, the present disclosure concerns a cell population of pluripotent stem cells and/or progenitor cells obtained by culturing the cells in the EMM of the present disclosure, such as in a medium comprising or consisting of: an aqueous sugar-free basal medium for mammalian cells, D-Galactose at a concentration sufficient for sustaining expansion of the cultured pluripotent stem cells, and a Stat3 activator, wherein said medium is free or substantially free of compounds that promote glycolytic metabolism. In one embodiment, the present disclosure concerns an in vitro cell culture comprising: a. mammalian pluripotent stem cells and/or progenitor cells; and b. a cell culture medium described herein. In some embodiments, the in vitro cell culture further comprises a matrix for supporting cells growth. In one embodiment, the present disclosure concerns an in vitro cell culture comprising in a culture vessel: a. mammalian pluripotent stem cells and/or progenitor cells; b. a matrix for supporting cells growth; and c. a cell culture medium described herein. The pluripotent stem cells and/or progenitor cells may be embryonic stem cells or induced pluripotent stem cells. In some embodiments, the pluripotent stem cells and/or progenitor cells are embryonic stem cells, induced pluripotent stem (iPS) cells, hepatic progenitor cells, cardiomyocyte progenitor cells, hypoblast stem cells and combinations thereof. In some embodiments, the pluripotent stem cells and/or progenitor cells are naïve extraembryonic endodermal cells. Stem cells may have the ability to undergo self-renewal and differentiation. In some embodiments, said pluripotent stem cells and/or progenitor cells are capable of enhanced multi-lineage differentiation. In some embodiments, the enhanced multi- lineage differentiation is tri-lineage differentiation. In some embodiments, the enhanced multi-lineage differentiation is tri-lineage differentiation, wherein the differentiation is
P6826PC00 tested using in vitro differentiation protocols and a panel of lineage markers to assess efficiency of differentiation. The pluripotent stem cells and/or progenitor cells may for example be derived from a primate. In some embodiments, the pluripotent stem cells and/or progenitor cells are primate pluripotent stem cells and/or progenitor cells. The source of the pluripotent stem cells and/or progenitor cell may for example be mammalian. In some embodiments, the pluripotent stem cells and/or progenitor cells are derived from a mammal, such as from a human, non-human primate, murine, pig, rat, horse, rabbit, sheep, guinea pig, gerbil, cattle, donkeys, goats, oxen, dogs and cats. In some embodiments, the pluripotent stem cells and/or progenitor cells are human embryonic stem cells. The cell cycle is the series of events that take place in a cell leading to its division and duplication (replication) that produces two daughter cells. Two major phases of the cell cycle are the S phase (DNA synthesis phase), in which DNA duplication occurs, and the M phase (mitosis), in which the chromosomes segregation and cell division occurs. The eukaryotic cell cycle is traditionally divided into four sequential phases: G1, S, G2, and M. G1, S, and G2 together can collectively be referred to as "interphase". Under certain conditions, cells can delay progress through G1 and can enter a specialized resting state known as G0 (G zero), in which they can remain for days, weeks, or even years before resuming proliferation. The period of transition from one state to another can be referred to using a hyphen, for example, G1/S, G2/M, etc. The present inventors have found that culturing pluripotent stem cells and/or progenitor cells in the EEM of the present disclosure results in a population of pluripotent stem cells and/or progenitor cells with a longer cell cycle compared to when the cells are cultured in a conventional medium, that is a medium comprising glucose as the main sugar and/or a medium comprising compounds that stimulate glycolysis. Thus, in some embodiments, the pluripotent stem cells and/or progenitor cells of the present disclosure have a cell cycle length of at least 48h after 3 or more passages. In some embodiments, the pluripotent stem cells and/or progenitor cells of the present disclosure have a cell cycle length of at least 48h after 1 or more passages, such as at least 48h after 2 or more passages, such as a cell cycle length of at least 48h after 3 or
P6826PC00 more passages, such as a cell cycle length of at least 48h after 4 or more passages, such as a cell cycle length of at least 48h after 5 or more passages, such as a cell cycle length of at least 48h after 6 or more passages, such as a cell cycle length of at least 48h after 7 or more passages, such as a cell cycle length of at least 48h after 8 or more passages, such as a cell cycle length of at least 48h after 9 or more passages, or such as a cell cycle length of at least 48h after 10 or more passages. In some embodiments, the pluripotent stem cells and/or progenitor cells of the present disclosure have a cell cycle length of at least 72h after 1 or more passages, such as at least 72h after 2 or more passages, such as a cell cycle length of at least 72h after 3 or more passages, such as a cell cycle length of at least 72h after 4 or more passages, such as a cell cycle length of at least 72h after 5 or more passages, such as a cell cycle length of at least 72h after 6 or more passages, such as a cell cycle length of at least 72h after 7 or more passages, such as a cell cycle length of at least 72h after 8 or more passages, such as a cell cycle length of at least 72h after 9 or more passages, or such as a cell cycle length of at least 72h after 10 or more passages. In some embodiments, the pluripotent stem cells and/or progenitor cells of the present disclosure have a cell cycle length of at least 96h after 1 or more passages, such as at least 96h after 2 or more passages, such as a cell cycle length of at least 96h after 3 or more passages, such as a cell cycle length of at least 96h after 4 or more passages, such as a cell cycle length of at least 96h after 5 or more passages, such as a cell cycle length of at least 96h after 6 or more passages, such as a cell cycle length of at least 96h after 7 or more passages, such as a cell cycle length of at least 96h after 8 or more passages, such as a cell cycle length of at least 96h after 9 or more passages, or such as a cell cycle length of at least 96h after 10 or more passages. In some embodiments, the pluripotent stem cells and/or progenitor cells of the present disclosure have a cell cycle length of 25h to 60h after 1 or more passages, such as of 25h to 60h after 2 or more passages, such as a cell cycle length of 25h to 60h after 3 or more passages, such as a cell cycle length of 25h to 60h after 4 or more passages, such as a cell cycle length of 25h to 60h after 5 or more passages, such as a cell cycle length of 25h to 60h after 6 or more passages, such as a cell cycle length of 25h to 60h after 7 or more passages, such as a cell cycle length of 25h to 60h after 8 or more passages, such as a cell cycle length of 25h to 60h after 9 or more passages, or such as a cell cycle length of 25h to 60h after 10 or more passages.
P6826PC00 In some embodiments, the pluripotent stem cells and/or progenitor cells of the present disclosure have a cell cycle length after 3 or more passages longer than when the same pluripotent stem cells and/or progenitor cells are cultured in a cell culture medium comprising glucose and/or promoting glycolysis. In some embodiments, the pluripotent stem cells and/or progenitor cells comprise a higher proportion of cells in G1 phase and lower population of cells in G2/M. In some embodiments, the proportion of cells in the G1 phase is between 35 and 55%, such as between 35% and 53%, between 35% and 51%, between 35% and 49%, between 35% and 47%, between 35% and 45%, between 35% and 43%, between 35% and 41%, between 35% and 39%, between 35% and 37%, between 37% and 55%, between 37% and 53%, between 37% and 51%, between 37% and 49%, between 37% and 47%, between 37% and 45%, between 37% and 43%, between 37% and 41%, between 37% and 39%, between 39% and 55%, between 39% and 53%, between 39% and 51%, between 39% and 49%, between 39% and 47%, between 39% and 45%, between 39% and 43%, between 39% and 41%, between 41% and 55%, between 41% and 53%, between 41% and 51%, between 41% and 49%, between 41% and 47%, between 41% and 45%, between 41% and 43%, between 43% and 55%, between 43% and 53%, between 43% and 51%, between 43% and 49%, between 43% and 47%, between 43% and 45%, between 45% and 55%, between 45% and 53%, between 45% and 51%, between 45% and 49%, between 45% and 47%, between 47% and 55%, between 47% and 53%, between 47% and 51%, between 47% and 49%, between 49% and 55%, between 49% and 53%, between 49% and 51%, between 51% and 55%, between 51% and 53%, or between 53% and 55%. In some embodiments, the proportion of cells in the G1 phase is about 43%. In some embodiments, the proportion of cells in the G1 phase is determined by Hoechst staining. In some embodiments, cell cycle profiles are determined by staining with Hoechst33342 and analyzed by Flow Cytometry. In some embodiments, the proportion of cells in the G1 phase is determined by Hoechst staining followed by Flow Cytometry. Cells can generate energy via two principle processes, glycolysis and oxidative phosphorylation (OXPHOS). Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), may be used as measures of Oxidative Phosphorylation (OXPHOS) and glycolysis, respectively. In some embodiments, the pluripotent stem cells and/or progenitor cells use oxidative phosphorylation (OXPHOS) to obtain energy
P6826PC00 to a larger extent than glycolysis. In some embodiments, the pluripotent stem cells and/or progenitor cells have a basal oxygen consumption rate to extracellular acidification rate (OCR:ECAR) ratio of at between 5:1 and 20:1, such as between 5:1 and 17:1, such as between 5:1 and 14:1, such as between 5:1 and 11:1, such as between 5:1 and 8:1, such as between 8:1 and 20:1, such as between 8:1 and 17:1, such as between 8:1 and 14:1, such as between 8:1 and 11:1, such as between 11:1 and 20:1, such as between 11:1 and 17:1, such as between 11:1 and 14:1, such as between 14:1 and 20:1, such as between 14:1 and 17:1, such as between 17:1 and 20:1. In some embodiments, the pluripotent stem cells and/or progenitor cells have a basal oxygen consumption rate to extracellular acidification rate (OCR:ECAR) ratio of at between 5:1 and 20:1. Cell pluripotency is a continuum, ranging from the completely pluripotent cell that can form every cell of the embryo proper, e.g., embryonic stem cells and iPSCs, to the incompletely or partially pluripotent cell that can form cells of all three germ layers but that may not exhibit all the characteristics of completely pluripotent cells, such as, for example, germline transmission or the ability to generate a whole organism. The pluripotency of cells may be analyzed by measuring pluripotency markers. The pluripotent stem cells and/or progenitor cells described herein may express one or more enhanced pluripotency markers, which may be specific markers of enhanced pluripotency of the cells. In some embodiments, the pluripotent stem cells and/or progenitor cells express one or more enhanced pluripotency markers. In some embodiments, the enhanced pluripotency markers are canonical epiblast markers and/or extra-embryonic (hypoblast or trophoblast) markers. In some embodiments, the enhanced pluripotency markers are Nanog, Hhex, Pdgfra, Klf4, Sox2, Tead1,Gata4, Sox7, Gata3, Tead4, and/or Krt18. In some embodiments, the pluripotent stem cells and/or progenitor cells express two or more, such as three or more such as all of the enhanced pluripotency markers selected from Nanog, Hhex, Pdgfra, Gata4, KLF4, Sox2, Tead1,4, Sox7, Gata3, Tead4, Krt18. In some embodiments, the pluripotent stem cells and/or progenitor cells have enhanced chromatin accessibility of embryonic- (ERK repressed) and/or extraembryonic- (ERK induced) enhancers within 24h of culturing compared to a population of pluripotent stem cells and/or progenitor cells cultured in a medium comprising glucose or other compounds that promote glycolysis.
P6826PC00 In some embodiments, the pluripotent stem cells and/or progenitor cells reduced acetylation of histone lysines compared to a population of pluripotent stem cells and/or progenitor cells cultured in a medium comprising glucose or other compounds that promote glycolysis. In some embodiments, the pluripotent stem cells and/or progenitor cells reduced acetylation of histone lysines at H3K9, H3K27 and/or H4K16 compared to a population of pluripotent stem cells and/or progenitor cells cultured in a medium comprising glucose or other compounds that promote glycolysis. In some embodiments, the pluripotent stem cells and/or progenitor cells comprise increased deacetylation of SOX2, OCT4, NANOG, TEAD4, YAP1, TFAP2C, and/or MYC compared to a population of pluripotent stem cells and/or progenitor cells cultured in a medium comprising glucose or other compounds that promote glycolysis. In some embodiments, the pluripotent stem cells and/or progenitor cells comprise changed levels of 2-Piperidinone, 3-(4-Hydroxy-3-methoxyphenyl)-2-methyllactic acid, 3-Indolepropionic acid, 3-Methoxybenzenepropanoic acid, 5-Hydroxydantrolene, 5- Methylcytidine, 5'-Methylthioadenosine, 7-Methylguanine, Adenine, Adenosine monophosphate, ADP, Benzoyl ecgonine, Beta-Alanine, Betaine, Beta-Leucine, Carbidopa, CDP, Choline, Citraconic acid, Creatine, Cytidine, Dimethadione, Dimethylglycine, Erythronic acid, Gamma-Butyrolactone, Gamma-Glutamylcysteine, Glucose , Glutathione, Guanosine, Guanosine monophosphate, Homogentisic acid, Homovanillic acid, Hypotaurine, Hypoxanthine, Inosine, Isosorbide Dinitrate, L- Acetylcarnitine, L-Arginine, L-Aspartic acid, L-Carnitine, L-Cysteine, L-Glutamic acid, L- Histidine, L-Isoleucine, L-Lactic acid, L-Lysine, L-Methionine, L-Norleucine, L- Phenylalanine, L-Proline, L-Serine, L-Threonine, L-Tryptophan, L-Tyrosine, L-Valine, Methsuximide, N-(2-Hydroxyethyl)-morpholine N-oxide, N(6)-Methyllysine, N4- Acetylsulfamethoxazole, N-Acetylaspartylglutamic acid, NADH, Niacinamide, NAD, Nicotinamide ribotide, Nitroxoline, Pantothenic acid, Phenylpyruvic acid, Phosphocholine, Phosphoric acid, Phosphoserine, Pindolol, Pipecolic acid, Pirbuterol, Pseudoephedrine, Pyridoxal, Pyridoxamine, Pyroglutamic acid, S- Adenosylhomocysteine, Sevoflurane, Succinyladenosine, Taurine, Uracil, Uridine 5'- diphosphate, Uridine 5'-monophosphate, and/or Vigabatrin compared to a population of
P6826PC00 pluripotent stem cells and/or progenitor cells cultured in a medium comprising glucose or other compounds that promote glycolysis. In some embodiments, the pluripotent stem cells and/or progenitor cells comprise reduced PARyalation activity of PARP enzymes compared to a population of pluripotent stem cells and/or progenitor cells cultured in a medium comprising glucose or other compounds that promote glycolysis. Method for maintaining, rejuvenating and/or promoting enhanced extra-embryonic competences. As described herein above, the present invention takes advantage of forcing a metabolic change in embryonic stem cells to promote a developmental identity that approximates the inner cell mass (ICM) of the early mammalian blastocyst in cultures, referred to as enhanced metabolic ESCs (EMESCs). Creation of EMESCs depends on inhibition of glycolysis and stimulation of oxidative phosphorylation (OXPHOS), that in turns activates NAD+-dependent deacetylases of the Sirtuin family. This is accomplished by utilization of Enhanced Metabolic Media (EMM) described herein, which stimulate an enhanced ICM-like metabolic signature in which cells elevate their levels of OXPHOS and reduce dependence on glycolysis by forcing extensive utilization of OXPHOS as a means to generate sufficient ATP for cell survival. The medium may thus be used in a method for maintaining, enhancing and/or promoting pluripotency, and/or for enhancing the potency of pluripotent cells. The medium may be used in a method for rejuvenating mammalian pluripotent stem cells and/or progenitor cells. The medium may be used in a method for promoting enhanced extra-embryonic competence of mammalian pluripotent stem cells and/or progenitor cells. The medium may be used in enhancing expression of extra-embryonic hypoblast or trophoblast markers, such as Gata6, Gata4, Sox7, Emp1, Col4a1, Sox17, Pdgfra, Tbx15, Foxq1, Marcks, Krt8, Krt18, Emoes, Dab2, Dusp4, Gata3, Tead4, Lrp2, Tagin2, Bmyc, Cldn2, and Tbx1. In one embodiment, the present disclose concerns a method for maintaining, enhancing and/or promoting pluripotency, and/or for enhancing the potency of pluripotent cells in a population of mammalian pluripotent stem cells and/or progenitor cells, the method comprising culturing the pluripotent stem cells and/or progenitor cells in a cell culture medium comprising or consisting of:
P6826PC00 aqueous sugar-free basal medium for mammalian cells supplemented with D-galactose at a concentration sufficient for sustaining expansion of the cultured pluripotent stem cells, and a Stat3 activator, wherein said medium is free or substantially free of compound that promotes glycolytic metabolism. As described above, the medium may be used in a method for rejuvenating mammalian pluripotent stem cells and/or progenitor cells, in particular ineffective or aged mammalian pluripotent stem cells and/or progenitor cells, that is cells that are characterized by reduced differentiation capacity. In one embodiment, the present disclose concerns a method for rejuvenating mammalian pluripotent stem cells and/or progenitor cells characterized by reduced differentiation capacity, the method comprising culturing the pluripotent stem cells and/or progenitor cells in a cell culture medium comprising or consisting of: aqueous sugar-free basal medium for mammalian cells supplemented with D-galactose at a concentration sufficient for sustaining expansion of the cultured pluripotent stem cells, and a Stat3 activator, wherein said medium is free or substantially free of compound that promotes glycolytic metabolism. In some embodiments, said reduced differentiation capacity arises from the age of the cells. In some embodiments, said pluripotent stem cells and/or progenitor cells characterized by reduced differentiation capacity are characterized as ineffective cells. In some embodiments, said reduced differentiation capacity is reduced pluripotency. In one embodiment, the present disclose concerns a method for promoting enhanced extra-embryonic competence of mammalian pluripotent stem cells and/or progenitor cells, the method comprising culturing the pluripotent stem cells and/or progenitor cells in a cell culture medium comprising or consisting of: aqueous sugar-free basal medium for mammalian cells supplemented with D-galactose at a concentration sufficient for sustaining expansion of the cultured pluripotent stem cells, and a Stat3 activator,
P6826PC00 wherein said medium is free or substantially free of compound that promotes glycolytic metabolism. In some embodiments, the cell culture medium is as defined herein. The pluripotent stem cells may for example be cultured on support matrixes, that for example may a mimic of an extracellular matrix. In some embodiments, the method comprises cultivating the pluripotent stem cells on a support matrix. In some embodiments, the support matrix may be gelatine, fibronectin, laminin, collagen, basement membrane matrix such as Matrigel, and/or mouse embryonic fibroblast feeder cells, and combinations thereof. The pluripotent stem cells and/or progenitor cells may be pluripotent stem cells and/or embryonic stem cells. In some embodiments, the pluripotent stem cells and/or progenitor cells are embryonic stem cells, induced pluripotent stem (iPS) cells, hepatic progenitor cells, cardiomyocyte progenitor cells, hypoblast stem cells, and combinations thereof. In some embodiments, the pluripotent stem cells and/or progenitor cells are naïve extraembryonic endoderm. The origin of the pluripotent stem cells and/or progenitor cells may be for example be mammalian. In some embodiments, the pluripotent stem cells and/or progenitor cells are primate pluripotent stem cells and/or progenitor cells. In some embodiments, the pluripotent stem cells and/or progenitor cells are derived from a mammal, such as from a human, non-human primate, murine, pig, rat, horse, rabbit, sheep, guinea pig, gerbil, cattle, donkeys, goats, oxen, dogs and cats. In some embodiments, the pluripotent stem cells and/or progenitor cells are human embryonic stem cells. The method described herein may be capable of maintaining the cultured population in an undifferentiated state for a number of rounds of subculturing. In some embodiments, the method is capable of maintaining the cultured population in an undifferentiated state for at least two passages, such as for at least three passages, such as for at least four passages, such as for at least five passages, such as for at least six passages, such as for at least seven passages, such as for at least eight passages, such as for at least nine passages, such as for at least ten passages.
P6826PC00 As described herein above, cells can generate energy via two principal processes, glycolysis and oxidative phosphorylation (OXPHOS). The method described herein may inhibit glycolysis and stimulate oxidative phosphorylation (OXPHOS). In some embodiments, the method inhibits glycolysis in the population of cultured pluripotent stem cells within three hours of culturing. In some embodiments, the method stimulates OXPHOS in the population of cultured pluripotent stem cells within three hours of culturing. The method described herein may enhance the expression of one or more pluripotency markers compared to a method comprising culturing cells in a medium comprising glucose or other compounds that promote glycolysis. In some embodiments, the method enhances expression of one or more pluripotency markers in the population of pluripotent stem cells within three hours of culturing. In some embodiments, the method enhances expression of one or more pluripotency markers in the population of pluripotent stem cells within three hours of culturing compared to a population of pluripotent stem cells cultured in a medium comprising glucose or other compounds that promote glycolysis. In some embodiments, the method enhances expression of one or more markers selected from Nanog, Hhex, PDGFRA, KLF4, Sox2, Tead1, Gata4, Sox7, Gata3, Tead4, Krt18 within three hours of culturing. In some embodiments, the method enhances chromatin accessibility of embryonic- (ERK repressed) and/or extraembryonic- (ERK induced) enhancers in the population of pluripotent stem cells within 24h of culturing compared to a population of pluripotent stem cells cultured in a medium comprising glucose or other compounds that promote glycolysis. In some embodiments, the method increases deacetylation of SOX2, OCT4, NANOG, TEAD4, YAP1, TFAP2C, and/or MYC in the population of pluripotent stem cells compared to a population of pluripotent stem cells cultured in a medium comprising glucose or other compounds that promote glycolysis. In some embodiments, the method changes levels of 2-Piperidinone, 3-(4-Hydroxy-3- methoxyphenyl)-2-methyllactic acid, 3-Indolepropionic acid, 3- Methoxybenzenepropanoic acid, 5-Hydroxydantrolene, 5-Methylcytidine, 5'- Methylthioadenosine, 7-Methylguanine, Adenine, Adenosine monophosphate, ADP,
P6826PC00 Benzoyl ecgonine, Beta-Alanine, Betaine, Beta-Leucine, Carbidopa, CDP, Choline, Citraconic acid, Creatine, Cytidine, Dimethadione, Dimethylglycine, Erythronic acid, Gamma-Butyrolactone, Gamma-Glutamylcysteine, Glucose , Glutathione, Guanosine, Guanosine monophosphate, Homogentisic acid, Homovanillic acid, Hypotaurine, Hypoxanthine, Inosine, Isosorbide Dinitrate, L-Acetylcarnitine, L-Arginine, L-Aspartic acid, L-Carnitine, L-Cysteine, L-Glutamic acid, L-Histidine, L-Isoleucine, L-Lactic acid, L-Lysine, L-Methionine, L-Norleucine, L-Phenylalanine, L-Proline, L-Serine, L- Threonine, L-Tryptophan, L-Tyrosine, L-Valine, Methsuximide, N-(2-Hydroxyethyl)- morpholine N-oxide, N(6)-Methyllysine, N4-Acetylsulfamethoxazole, N- Acetylaspartylglutamic acid, NADH, Niacinamide, NAD, Nicotinamide ribotide, Nitroxoline, Pantothenic acid, Phenylpyruvic acid, Phosphocholine, Phosphoric acid, Phosphoserine, Pindolol, Pipecolic acid, Pirbuterol, Pseudoephedrine, Pyridoxal, Pyridoxamine, Pyroglutamic acid, S-Adenosylhomocysteine, Sevoflurane, Succinyladenosine, Taurine, Uracil, Uridine 5'-diphosphate, Uridine 5'-monophosphate, and/or Vigabatrin in the population of pluripotent stem cells compared to a population of pluripotent stem cells cultured in a medium comprising glucose or other compounds that promote glycolysis. The method described herein may for example be used for obtaining homogenous undifferentiated colonies or higher proportion of cells in the G1 phase. In some embodiments, the method comprises culturing the pluripotent stem cells for at least two passages. In some embodiments, the method comprises culturing the pluripotent stem cells for at least two passages thereby obtaining colonies of homogeneously undifferentiated pluripotent stem cells after two or more passages. In some embodiments, the method comprises culturing the pluripotent stem cells for at least two passages thereby obtaining a population of cells comprising a higher proportion of cells in G1 phase and lower population of cells in G2/M after 2 or more passages. Methods for culturing embryos, gametes, stem cells, blastoids In vitro models of mouse blastocysts, so-called blastoids have been generated using two different approaches: (1) by assembling different blastocyst-like cells together including mouse embryonic stem cells (ESCs) with trophoblast stem cells (TSCs); ESCs, TSCs, and extraembryonic endoderm (XEN) stem cells; extended (or expanded) pluripotent stem cells (EPSCs) with TSCs or by (2) differentiating EPSCs into blastocyst- like structures.
P6826PC00 The present disclosure relates to the novel feature is that the blastoids obtained in the medium of the present disclosure have a higher proportion of hypoblast cells compared to blastoids obtained in a traditional, glucose-based cell culture medium. Hypoblast cells can for example be identified because they are GATA4+. Blastoids are known to have a low proportion of hypoblast, in particular they are known to have a substantially lower proportion of hypoblast compared to embryos. Surprisingly, blastoids obtained according to the present disclosure, that is by culturing a cell population of: naïve embryonic stem cells (ESc), naïve induced pluripotent stem cells (iPSs), and/or extra-embryonic endoderm cells in a cell culture medium comprising or consisting of: an aqueous sugar-free basal medium for mammalian cells, D-Galactose at a concentration sufficient for sustaining expansion of the cultured pluripotent stem cells, and a Stat3 activator, wherein said medium is free or substantially free of compounds that promote glycolytic metabolism, have a proportion of hypoblast that is comparable or closer to the physiological proportion of hypoblast cells normally found in embryos. In one embodiment, the present disclosure concerns a method of producing a blastocyst-like structure, the method comprising: a) obtaining a cell population of: naïve embryonic stem cells (ESc), naïve induced pluripotent stem cells (iPSs), and/or extra-embryonic endoderm cells; and b) culturing said cell population in a cell culture medium comprising or consisting of: an aqueous sugar-free basal medium for mammalian cells, D-Galactose at a concentration sufficient for sustaining expansion of the cultured pluripotent stem cells, and a Stat3 activator, wherein said medium is free or substantially free of compounds that promote glycolytic metabolism, thereby producing a blastocyst-like structure characterized by comprising a higher proportion of hypoblast cells compared to a blastocyst-like structure obtained in a glucose-based cell culture medium.
P6826PC00 In one embodiment, the blastocyst-like structure characterized by comprising a higher proportion of hypoblast cells compared to a blastocyst-like structure obtained in a glucose-based cell culture medium is a blastocyst-like structure characterized by comprising a high proportion of GATA-positive (GATA+) cells, or a higher proportion of GATA+ cells compared to a blastocyst-like structure obtained in a glucose-based cell culture medium. In one embodiment, the cell population of: naïve embryonic stem cells (ESc), naïve induced pluripotent stem cells (iPSs), and/or extra-embryonic endoderm cells, is cultured in a cell culture medium disclosed herein. In one embodiment, the present disclose concerns a blastocyst-like structure obtained by the method as described herein, wherein said blastocyst-like structure is characterized by comprising a high proportion of hypoblast cells, such as a high proportion of GATA4+ cells. In one embodiment, the present disclose concerns a blastocyst-like structure obtained by the method as described herein, wherein said blastocyst-like structure is characterized by comprising a higher proportion of hypoblast cells compared to a blastocyst-like structure obtained in a glucose-based cell culture medium. In one embodiment, the present disclose concerns a blastocyst-like structure characterized by comprising a high proportion of hypoblast cells, such as a high proportion of GATA4+ cells. In one embodiment, the present disclose concerns a blastocyst-like structure characterized by comprising a higher proportion of hypoblast cells, such as a higher proportion of GATA4+ cells compared to a blastocyst-like structure obtained in a glucose-based cell culture medium. Many mammals can temporally uncouple conception from parturition by pacing down their development around the blastocyst stage.
P6826PC00 The blastocysts may enter a state of dormancy, called diapause, as described by Iyer et al 2024. In some embodiments, said blastocyst-like structure of the present disclosure has a diapause-like phenotype. The cell culture medium and the methods disclosed herein may be helpful in assisted reproduction. In one embodiment, the present disclosure concerns a method for handling and/or manipulating and/or culturing an embryo for assisted reproduction, a gamete or a stem cell, the method comprising handling and/or manipulating and/or culturing the embryo for assisted reproduction or gamete or stem cell in a culture medium comprising or consisting of: an aqueous sugar-free basal medium for mammalian cells, D-Galactose at a concentration sufficient for sustaining expansion of the cultured pluripotent stem cells, and a Stat3 activator, wherein said medium is free or substantially free of compounds that promote glycolytic metabolism. For example, a stem cell can be an embryonic stem cell, an adult stem cell and induced pluripotent stem cell. In one embodiment, handling and/or manipulating and/or culturing the embryo for assisted reproduction or gamete or stem cell in a cell culture medium of the present disclosure improves the development of an embryo. For example, it improves the development of an embryo by increasing the proportion of hypoblast cells in said embryo, gamete or stem cell compared to when said embryo, gamete or stem cell are cultured in a cell culture medium comprising glucose. In one embodiment, handling and/or manipulating and/or culturing the embryo for assisted reproduction or gamete or stem cell in a cell culture medium of the present disclosure comprises increasing the proportion of hypoblast cells in said embryo, gamete or stem cell.
P6826PC00 In one embodiment, the embryo, gamete or stem cell is a mammalian embryo, gamete or stem cell. In one embodiment, the embryo, gamete or stem cell is a human embryo, gamete or stem cell. In one embodiment, the embryo, gamete or stem cell are derived from a mammal, such as from a human, non-human primate, murine, pig, rat, horse, rabbit, sheep, guinea pig, gerbil, cattle, donkeys, goats, oxen, dogs and cats. In one embodiment, the embryo, gamete or stem cell is cultured individually. For example, in the context of assisted reproduction. In one embodiment, the embryo is cultured to the blastocyst stage. For example, in the context of assisted reproduction. Examples Example 1. Altering the balance between OXPHOS and Glycolysis to reprogram ESCs to an early embryonic state Methods Cell culture ESC lines were maintained in complete ESC medium: DMEM no glucose (Gibco #11966025) supplemented with 10% FBS (Gibco), 4.5g/L D-glucose (Sigma-Aldrich), 100 µM 2-mercaptoethanol (Sigma-Aldrich), 100mM MEM nonessential amino acids, 2mM L-glutamine, 1 mM sodium pyruvate (all from Gibco), and 1,000 units/ml LIF (made in house) on gelatinized tissue culture flasks (Corning). EMM is as described above, but without the addition of glucose and pyruvate, and with 4.5g/L D-galactose. For 2i/LIF and KOSR/LIF culture, ESCs were maintained as previously described in Martin Gonzalez et al.2016. Seahorse Assays ESCs were seeded into 96-well Seahorse plates precoated with gelatine at 40x104 cells per well. Oxygen Consumption Rate (OCR) and extracellular acidification rate (ECAR) were determined by the XF cell mito-stress test (#101706-100, Seahorse Biosciences) as described previously Mahato et al 2014, and basal measurements
P6826PC00 were taken from the third recorded time point. Values were normalized to total protein levels per well. Fluorescence Activated Cell Sorting (FACS) and Flow Cytometry Cells were dissociated with Accutase (A6964, Sigma) and incubated with the appropriate antibody in 10% FCS:PBS for 30 min, washed extensively, and analyzed on an LSR Fortessa (BD Biosciences). Dead cells were excluded based on DAPI inclusion. For FACS followed by Seahorse assays, Hhex-Venus ESCs were stained for PECAM-1 as described previously (Canham et al., 2010), and sorted with a BD FACS Aria III cell sorter. Cells were collected in 10% FCS:PBS, then reconstituted in Serum/LIF, 2i/LIF, or KOSR/LIF and seeded in Seahorse plates, as described above. For flow cytometry, cells were treated as above and analyzed using a BD LSR Fortessa. Data was analyzed with the FACSDiva and FCS Express 6 software (BD Biosciences). Immunofluorescence Cells were washed and fixed in 4% formaldehyde (Fisher Scientific, PI-28906), blocked, and permeabilized in 5% donkey serum and 0.3% Triton. Antibodies were incubated overnight in 1% BSA and 0.3% Triton in PBS and subsequently visualized with the appropriate secondary antibody (Alexa Fluor, Molecular Probes) and imaged using a Leica TCS SP8 confocal microscope and analyzed using Imarisx649.5.1 software. Alkaline Phosphatase Staining ESCs were plated at clonal density and cultured for 9–10 days. Alkaline phosphatase staining was carried out with the diagnostic kit 86-R (Sigma) as per manufacturer’s instructions. Colonies were scored as 100% differentiated or undifferentiated or as mixed colonies containing both undifferentiated and differentiated cells. Colonies were imaged using a Nikon AZ-100 microscope and quantified using ImageJ. RNA-sequencing was conducted as described in Example 3. Results Having established that ICM-like ESC sub-populations exhibit higher rates of OXPHOS
P6826PC00 than pluripotent ESCs, it was assessed whether forcing an increased dependence on OXPHOS relative to glycolysis would be sufficient to induce this cell state. To achieve this, the inventors replaced D-glucose and pyruvate in Serum/LIF media with D- galactose. The inventors refer to this media as Enhanced Metabolic Media (EMM), as it should stimulate an enhanced ICM-like metabolic signature in which cells elevate their levels of OXPHOS and reduce dependence on glycolysis. Within 3h of replacing standard ESC media with EMM, the inventors observed a rapid simultaneous inhibition of glycolysis and stimulation of OXPHOS (Fig.1a). This state was stabilized at 24h, and ESCs could be maintained in this culture condition. Culture of ESCs in EMM was found to promote expression of fluorescent reporters for both the Epi/pluripotency marker Nanog and PrE marker Hhex. Flow cytometry indicated that expression of both factors was elevated and more uniform in EMM, suggesting the induction of an ICM-like state. Levels of Nanog-eGFP were both high and homogenous, with a marked reduction of the Nanoglow ESC subpopulation (Fig. 1b). In addition, the entire population of ESCs expressed higher and more homogeneous levels of the Hhex-Venus reporter (Fig.1c) and elevated expression of both NANOG protein and Hhex-Venus were present within the same cells (Fig.1d-e). Pluripotency markers NANOG, OCT4 and SOX2 were also co-expressed with the PrE transcription factor (TF) GATA6 in a small population of EMESCs, suggesting these GATA6+ cells represented an unsegregated ICM-like state rather than the spontaneous PrE differentiation observed Serum/LIF conditions (Fig.1f-g). The inventors also observed no significant increase in apoptosis in the Nanoglow population (Fig.1h-i), suggesting that altering the relative utilization of OXPHOS and glycolysis induced a more ICM-like gene expression state, rather than selecting for it. The inventors found that the functional properties of EMESCs also reflected their enhanced ICM-like phenotype. Colonies expanded in EMM were homogeneously undifferentiated (Fig.1j and k), and consistent with the smaller size of EMM colonies, the inventors observed that ESCs grown in these conditions had a considerably longer cell cycle length of about 48h or more, proliferating at roughly half the rate of Serum/LIF-cultured ESCs, with a significantly increased proportion of cells in G1 phase and less in G2/M (Fig.1l) as has been reported for 2i/LIF culture.
P6826PC00 To assess the impact EMM over longer culture periods, EMESCs were cultured for 10 and 20 passages, alongside ESCs cultured in Serum/LIF and 2i/LIF for the same time period. The phenotype of EMESCs after 10 passages was similar to that of serum/LIF cultured ESCs (Fig.1m). The investigators also performed RNA-seq on these long term cultures, and found that EMESCs express fewer apoptotic genes than ESCs cultured in 2i/LIF after 10 and 20 passages, indicating their long-term stability (Fig 1n- p), while embryonic (pluripotency) (Fig.1q-s) and extra-embryonic genes (Fig 1t-v) were maintained at similar levels. Example 2. EMM culture affects embryonic development Aim: To examine whether EMM culture affected embryonic development, the inventors cultured mouse embryos from zygotes to late-stage blastocysts ex vivo, in a galactose- containing medium similar to EMM. Methods Embryo culture Oocytes were collected from the oviducts of prepubescent hormone-stimulated C57BL/6NRj females and fertilized in vitro according to CARD protocols, adapted by Infrafrontier (infrafrontier.eu/knowledgebase/protocols/cryopreservation-protocols). The resulting zygotes were cultured in KSOM medium (made in house), which contained either D-glucose or D-galactose. These embryos were then cultured for 5 days to reach the equivalent of an E4.5 in vivo embryo. Embryos were cultured in distinct microdrops for each condition, overlaid with embryo culture mineral oil (Sigma). Embryos were cultured at 37°C, 5% CO2 and 90% relative humidity. Embryos were then stained for NANOG (Epi marker) and CDX2 (TE marker) to analyze any differences between conditions. Animal work was carried in accordance with European legislation and was authorized by and carried out under Project License 2018-15-0201-01520 issued by the Danish Regulatory Authority. Mice were maintained in a 12-h light/dark cycle in the designated facilities at the University of Copenhagen, Denmark. Morula injection and E6.5 contribution E2.5 morula embryos were de-compacted in PB1 medium without calcium and magnesium for 20 min at room temperature and 5 H2B-Tomato tagged ESCs, previously cultured in EMM, Serum/LIF or 2i/LIF, were introduced by microinjection. Resultant embryos were transferred to E0.5 pseudo-pregnant mothers. At E6.5,
P6826PC00 embryos were dissected from the decidua and contribution of H2B-Tomato ESCs was assessed. Embryos were stained by immunofluorescence for GATA6 (Visceral and Parietal endoderm marker) and KRT7 (Trophectoderm marker) as described in Example 1. Results: After culture, most embryos cultured in KSOM+galactose had developed into normal blastocysts that exhibited higher numbers of NANOG positive central cells, suggesting an expanded ICM (Fig.2a). To determine whether EMESCs retained pluripotency, the inventors tested their capacity to colonize specific lineages in chimeras. ESCs constitutively expressing a H2B-Tomato fluorescent reporter were cultured in Serum/LIF, EMM, or 2i/LIF media and then injected into wild-type morulae. The inventors found that the EMESCs were extremely efficient at generating chimeras, giving higher levels of epiblast contribution than either 2i/LIF or Serum/LIF cultured ESCs (33% for Serum/LIF, 70% for EMM, 50% for 2i/LIF) (Fig.2b-c). Based on both position and whole-mount immunostaining for GATA6 and trophoblast marker KRT7, the inventors also observed contribution to both extra-embryonic lineages at similar levels to what the inventors observed for 2i/LIF (GATA6: 0% for Serum/LIF, 10% for EMM, 2.94% for 2i/LIF; KRT7: 0% for Serum/LIF, 5% for EMM, 2.94% FOR 2i/LIF) (Fig.2c) Example 3. Early Transcriptional and Chromatin Accessibility Changes in Response to Culture in EMM. Aim: As EMM induced ICM-like phenotypes in EMESCs, the inventors sought to compare the transcriptome of EMM-cultured ESCs with mouse pre-implantation embryos from different developmental stages. Methods RNA-seq, ATAC-seq and Bioinformatics Total RNA was purified by standard methods and rRNA depleted using the Ribo-Zero kit (Illumina, as per the manufacturer’s instructions). Libraries were prepared for Illumina sequencing using the NEBNext Ultra kit as per manufacturer’s instructions. For all conditions, three biological replicate samples were collected from independent experiments. RNA-seq libraries were prepared on-bead using the NEBNext Ultra kit as
P6826PC00 per manufacturer’s instructions and subsequently sequenced using a Next-Seq 500 Sequencer (Illumina). Sequencing reads (60 bases) were aligned using the STAR package. Allocation of reads at introns (and exons) were examined using Table Browser (UCSC) to define the corresponding genomic intervals. Reads per gene per class were counted using HTSeq with the categorised alignment files as input. Genes were considered significantly regulated if they exhibited Log2FC>1, and Padj<0.01. ATAC-seq was performed following methods previously described in Buenrostro et al. 2013. Adherent cells were treated with Accutase to obtain a single cell suspension. Cells were counted and resuspended to obtain 50,000 cells per sample in ice-cold PBS. Cells were pelleted and resuspended in lysis buffer (10mM Tris-HCl pH 7.4, 10mM NaCl, 3mM MgCl2, 0.1% IGEPAL). Following a 10min centrifugation at 4°C, nucleic extracts were resuspended in transposition buffer for 30min at 37°C and purified using a QIAGEN MinElute PCR Purification kit following manufacturer’s instructions. Transposed DNA was eluted in a 10mL volume and amplified by PCR with Nextera primers (Buenrostro et al.2013) to generate paired-indexed libraries. A maximum of 12 cycles of PCR was used to prevent saturation biases based on optimization experiments performed using RT-qPCR. Library quality control was carried out using the Bioanalyzer High-Sensitivity DNA analysis kit. Libraries were sequenced as paired-end 50bp reads, sequenced using a Next-Seq 500 Sequencer (Illumina). For all conditions, two biological replicate samples were collected from independent experiments. ATAC regions were considered significantly regulated if they exhibited Log2FC>1, and Padj<0.01. Metaprofiles were generated from bigWig files using deepTools software (Ramírez et al.2014). Enhancer gene association was performed using GREAT (http://great.stanford.edu/). Data processing and analysis were performed using Computerome, the National Life Science Supercomputer at DTU (www.computerome.dk), and the Bioconductor package, DESeq2 (Love et al.2014). The IMAGE pipeline was performed to identify active motifs from the ATACseq and RNAseq data. The pipeline was obtained from Grud et al.2018, and is also available on GitHub (https://github.com/JesperGrud/IMAGE). The IMAGE pipeline required as input: the ATACseq data normalized by library size, the RNAseq raw count matrix and the primary assembly genome of mm10 from GENCODE in fasta format. Pipeline
P6826PC00 results were processed using custom scripts derived from the IMAGE pipeline GitHub (https://github.com/JesperGrud/IMAGE). Results While Serum/LIF-cultured ESCs were closer to the traditional naïve ESCs in this dataset, increased time in EMM shifted the transcriptome of EMM-cultured ESCs closer to that of the ICM (data not shown). Accordingly, the transcription of genes that characterize the ICM state were largely upregulated upon EMM culture, while genes representing the 2-cell state were unchanged. While genes of the early trophoblast were also upregulated, the inventors found that Cdx2, Elf5, and Tmem54, which characterize the differentiated trophoblast, were downregulated. When the transcriptomes of EMM-cultured ESCs were compared with those of other ESCs representing different states of pluripotency, EMM-cultured ESCs at 24h expressed more marker genes of naïve pluripotency, and fewer marker genes of primed pluripotency, compared with Serum-cultured ESCs (data not shown). The inventors found that the accessibility of both embryonic- (ERK repressed) or extraembryonic- (ERK induced) enhancers was increased following 24h in EMM (data not shown). The inventors found a general increase in accessibility of the complete set of ESC enhancers (Hamilton et al.2019) and the highly cooperative large enhancer sequences known as super enhancers, (SEs) (Whyte et al.2013) (data not shown). The inventors also observed a positive correlation between RNA expression and enhancer accessibility after 24h EMM culture (Fig.3). Conclusion: The inventors conclude that culture in EMM rapidly induces increased accessibility of enhancers associated with pre-implantation development, while shutting down those associated with later embryonic differentiation. Example 4. Role for NAD+-dependent Sirtuin activity in inducing EMESC phenotypes Aim: To connect the transcriptional response observed in EMM-cultured ESCs to metabolic change. Materials and methods: Western Blotting
P6826PC00 Blotting was performed as previously described in Hamilton et al.2013 except that primary antibodies were detected using fluorescently conjugated secondary antibodies (Alexa Fluor, Molecular Probes), visualized using a Chemidoc MP (Bio-Rad) and quantified using ImageJ. Real-Time qPCR Total RNA was collected using either Trizol (Invitrogen) or the RNeasy Mini Kit (QIAGEN). Genomic DNA was eliminated by DNase treatment (QIAGEN), and 1 μg of total RNA was used for first-strand synthesis with SuperScript III reverse transcriptase according to the manufacturer’s instructions. cDNA corresponding to 10ng total RNA was used for real-time (RT)-qPCR analysis using the Roche LC480, and target amplification was detected with the Universal Probe Library system. Values were normalized to the geometric mean for GAPDH, Pgk1 and Sdha expression. Immunostaining Immunostaining was performed as described in Example 1. RNA-Seq RNA-Seq was performed as described in Example 3. Flow cytometry Flow cytometry was performed as described in Example 1. Metabolome Cells (500,000 per sample) were cultured in Serum/LIF or EMM for 3h, 9h and 24h, then spun down at 500g for 3mins, aspirated and resuspended in 1ml 0.9% NaCl. Samples were spun down and aspirated again, then stored at -80°C until further processing. Metabolite extraction from the frozen pellets was then performed by adding 200µl pre-chilled 50%MeOH with 0.0007mg/ml D5-tryptophan, followed by sonication (5°C for 2 rounds, 30s each) and centrifugation (0°C at 15,000g for 10min).150µl supernatant was collected and frozen at -80°C, with 10µl from each sample collected in one tube to make a Quality Control (QC). Pellets were reconstituted with 250µl MilliQ H2O, then sonicated and the protein concentration was measured using the Bicinchoninic acid (BCA) assay (Pierce).
P6826PC00 LC-MS analysis was performed as described before by Dall et al., 2018, with few modification of the A and B solvent composition, injection volume and MSMS analysis. Briefly, metabolite extracts, QC samples and blanks were defrosted on ice, vortexed and set in a pre-chilled vial. Samples were maintained at 4 °C throughout the analysis. Chromatographic separation was performed using UHPLC Dionex Ultimate 3000 (Thermo Scientific, Germany) with Luna Polar C18 column (1.6 μm, 2.1 × 100 mm, Phenomenex, USA) with EVO C18 guard column (sub-2μm, 2.1 mm, Phenomenex, USA) kept at 40 °C. Solvent A and B were 0.1% formic acid in acetonitrile and 0.1% formic acid in LC-MS grade water, respectively. A flow rate of 0.3 mL/min was applied with a gradient elution profile: 95% B 0–1 min, 95%–5% B 1.0–10.0 min, 5% B 10.0– 12.0 min, 5–95% B 12.0–12.5 min, 95% B 12.5-14-5 min. LC was coupled with QToF Impact II mass spectrometer (Bruker Daltonics, Germany). Samples were analyzed in positive and negative mode.10 μL of the extract was injected in positive mode and 20 μL in the negative mode. MS spectra were acquired in the mass range 50–1000 mass to charge ratio (m/z) at 2.00 Hz spectra rate using the source settings for positive mode: absolute threshold 50 cts per 1000 sum, End Plate Offset 500 V, Capillary 4500 V, Nebulizer 2.0 Bar, Dry Gas 10.0 l/min, Dry Temperature 220 °C; Transfer: Funnel 1RF 150.0 Vpp, Funnel 2FR 200.0 Vpp, isCID Eergy 0.0 eV, Hexapole RF 50.0Vpp; Quadrupole: Ion Enegry 4.0 eV, Low Mass 100.0 m/z; Collision Cell: Collision Energy 7.0 eV, Transfer Time 65.0 μs, Collision RF 650.0 Vpp, Pre Pulse Storage 5.0 μs. In negative mode Capillary voltage was set to 3000 and other parameters were identical as described above for both modes. MSMS analysis of the QC samples for metabolite identification purposes was performed at the same LC-MS settings as the MS scans and collision energy set to 25 for both negative and positive mode. Raw data is available through MetaboLights platform: www.ebi.ac.uk/metabolights/MTBLS2691 (identification number of the study MTBLS2691) (Haug et al.2020). Raw data from the positive and negative mode were analysed separately using MetaboScape 4.0 (Vasilopoulou et al.2020) (Bruker Daltonics, Germany). Mass calibration was based on sodium formate clusters and lock- mass calibration with hexakis(2,2-difluoroethoxy)phosphazene (Apollo Scientific Ltd, UK). Feature detection was performed using an intensity threshold of 1000 counts in the positive mode and 500 in the negative. Metabolites annotation of detected molecular features with assigned MS/MS spectra was performed using HMDB Metabolite Library and MetaboBASE Personal MSMS library (Bruker Daltonics,
P6826PC00 Germany). Identified compounds were inspected manually for peak shape, retention time and structure. Final data was normalised by the intensity of internal standard within each sample and multiplied by its average value in the samples. Molecular feature was retained in the final data, if its intensity was over double compared to the blanks injections and its reproducibility was within 30% of CV in QC samples. Data was analysed using Metaboanalyst 2.0 software (Chong et al.2019). Results: The inventors quantified the relative abundance of metabolites in ESCs cultured in either Serum/LIF or EMM at multiple time points over a 24-hour period (Fig.4a, b).The inventors identified 85 metabolites that were differentially expressed between the two media conditions at all time points (data not shown). The metabolites that were differentially expressed between the two media conditions at all time points were 2- Piperidinone, 3-(4-Hydroxy-3-methoxyphenyl)-2-methyllactic acid, 3-Indolepropionic acid, 3-Methoxybenzenepropanoic acid, 5-Hydroxydantrolene, 5-Methylcytidine, 5'- Methylthioadenosine, 7-Methylguanine, Adenine, Adenosine monophosphate, ADP, Benzoyl ecgonine, Beta-Alanine, Betaine, Beta-Leucine, Carbidopa, CDP, Choline, Citraconic acid, Creatine, Cytidine, Dimethadione, Dimethylglycine, Erythronic acid, Gamma-Butyrolactone, Gamma-Glutamylcysteine, Glucose , Glutathione, Guanosine, Guanosine monophosphate, Homogentisic acid, Homovanillic acid, Hypotaurine, Hypoxanthine, Inosine, Isosorbide Dinitrate, L-Acetylcarnitine, L-Arginine, L-Aspartic acid, L-Carnitine, L-Cysteine, L-Glutamic acid, L-Histidine, L-Isoleucine, L-Lactic acid, L-Lysine, L-Methionine, L-Norleucine, L-Phenylalanine, L-Proline, L-Serine, L- Threonine, L-Tryptophan, L-Tyrosine, L-Valine, Methsuximide, N-(2-Hydroxyethyl)- morpholine N-oxide, N(6)-Methyllysine, N4-Acetylsulfamethoxazole, N- Acetylaspartylglutamic acid, NADH, Niacinamide, NAD, Nicotinamide ribotide, Nitroxoline, Pantothenic acid, Phenylpyruvic acid, Phosphocholine, Phosphoric acid, Phosphoserine, Pindolol, Pipecolic acid, Pirbuterol, Pseudoephedrine, Pyridoxal, Pyridoxamine, Pyroglutamic acid, S-Adenosylhomocysteine, Sevoflurane, Succinyladenosine, Taurine, Uracil, Uridine 5'-diphosphate, Uridine 5'-monophosphate, and Vigabatrin. At the 3h time point the inventors observed induction of L-Carnitine, with a reciprocal reduction in L-Acetylcarnitine (Fig.4c, d), indicating that ESCs are rapidly adapting to glucose depletion and catabolizing other molecules, such as fatty acids and amino acids, in order to generate intermediate metabolites for the tricarboxylic acid (TCA) cycle. Consistent with the need to engage fatty acid
P6826PC00 metabolism via the TCA cycle, the inventors found that culturing ESCs in EMM elevated levels of the mitochondrial membrane fatty acid transporter CPT1A by immunofluorescence (Fig.4e) and in the EMM RNA-seq dataset (Fig.4f). Pathway enrichment analysis of the differentially regulated metabolites revealed that in addition to alterations in individual amino acid metabolism, “Nicotinate and Nicotinamide Metabolism” was highly enriched (data not shown). 10/14 top significantly enriched metabolic pathways are also associated with NAD+ homeostasis. The inventors found that PARyalation activity of PARP enzymes was drastically reduced in EMM culture after 24h (Fig.4g) and that PARP inhibitors could not block EMESC induction by EMM (Fig. 4h), suggesting that immediate early impact of NAD+ on transcription is likely due to Sirtuin activity. Sirtuins are NAD+ dependent Class III Histone deacetylases (HDACs) and the inventors observed that EMM-cultured ESCs have reduced acetylation of histone lysines at H3K9, H3K27 and H4K16 after 24h (Fig.4i and k). Sirtuins (Sirt1-7) are dependent upon NAD+, converting it to nicotinamide (NAM) during histone deacetylation (Guarente et al.2011). Sirtuin-mediated deacetylation of histones and TFs has been linked to both somatic and ESC self-renewal, differentiation, and reprogramming (Williams et al.2016; Ryall et al. 2015). The inventors found that the transition of ESCs to ICM-like EMESCs induced by EMM was inhibited by the potent Sirtuin inhibitor NAM. Addition of NAM blocked the capacity of EMM to induce the Nanog-eGFP high population (Fig.4j), influence EMESC early gene expression (Fig. 4l), and promote the co-expression of NANOG within the GATA6-positive cells in EMESC culture (Fig. 4m-n). The inventors also observed that the addition of NAM to EMM restored acetylation of H3K9, H3K27 and H4K16 back to Serum/LIF-cultured ESC levels (Fig.4i). The inventors used an inhibitor for the broad-acting histone acetylases CREBBP (CBP) and EP300 (A-485, Lasko et al., 2017). The inventors found that transcription of Nanog was reduced to a similar degree in both Serum/LIF- and EMM-cultured ESCs after 3h (Fig.4o), indicating that a specific pattern of deacetylation regulated by Sirtuin proteins is responsible for the EMESC phenotype induced by EMM. Example 5. Deacetylation of ICM-specific TFs mediates enhancer occupancy in EMESCs Aim: To identify the targets of Sirtuin dependent deacetylation in EMM Materials and methods:
P6826PC00 Immunoprecipitation For immunoprecipitation ESCs were cultured in Serum/LIF, EMM or EMM+NAM for 24h, washed in ice-cold PBS, lysed in Young lysis buffer 1 (YB1: 50mM HEPES KOH, pH 7.5, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP-40 and 0.25% Triton X-100, supplemented with 1xcOmpleteTM EDTA-free protease inhibitor tablets (Roche) and 10mM Sodium Butyrate), then sonicated. Immunoprecipitation was performed against 10μg protein with 1μg antibody in YB1. Antibody–protein complexes were collected with the appropriate magnetic bead and extensively washed in YB1 and eluted in 1xLaemmli buffer. Samples were then analysed by Western blot as described in example 4. CUT&Tag CUT&Tag was performed as previously described (Kaya-Okur et al., 2019) with slight alterations. antibody incubation was done at 4°C overnight, Tn5 was purchased from EMBL Heidelberg and DNA precipitation was performed over the weekend at -80°C. DNA was amplified with 12 PCR cycles and the samples were sequenced paired end on an Illumina NextSeq 500. Reads were aligned to the mm10 primary assembly using Bowtie2 (Langmead and Salzberg, 2012). Reads were quality filtered using Samtools (Danecek et al., 2012) and duplicates marked and removed using Picard (Picard Toolkit.” 2019. Broad Institute, GitHub Repository. https://broadinstitute.github.io/picard/). Fragment bedgraphs were produced using DeepTools bamCoverage (Ramirez, 2016). Peak calling was performed using SEACR (Meers, 2019). Peak calling by Sparse Enrichment Analysis for CUT&RUN chromatin profiling. SEACR relaxed was used for for transcription factors and SEACR stringent was used for histone marks, using normalized mode with condition-matched IgG samples as controls. Peaks called in 2/3 replicates for Tead1 and Klf4, and in 4/6 replicates over two experiments for Sox2 were used. Active enhancers were defined as regions with called peaks for both H3K27ac and H3K4me1 in all replicates. Read fragments at ± 200 bp regions centered on enhancer summits were counted using Bedtools map (Quinlan and Hall, 2010). Proteome and Acetylome Sample Preparation: For proteome analysis, four mESC lines (E14-early passage (ep), E14-late passages (lp), E14-ZscancE, E14-FUCCI) served as biological replicates and were each cultured in either Serum/LIF (SL), 2iLIF (N2B27, Chiron, PD03, LIF), or KOSR/LIF (Gibco) for more than three passages. Cell pellets were collected and
P6826PC00 frozen at -20° C. For lysis, pellets were thawed on ice and resuspended in 6M Guanidinium Hydrochloride (GndCl) with 5mM TCEP, 10mM CAA, 100mM Tris pH 8.5 and heated to 99°C for 10 minutes. Lysates were sonicated and then digested with LysC (Wako), (enzyme:protein ratio of 1:100 (w/w)) for 2h at room temperature (RT), followed by dilution with 25mM Tris pH8.5 to 2M GndCl and further digested overnight with trypsin (Sigma:Aldrich) at 1:100 (w/w) at 37° C. Peptides were basified to 40 mM ammonium hydroxide then purified by StageTip (C18 material) high pH fractionation. For this, StageTips were first activated in 100% methanol, then equilibrated in 80% acetonitrile in 50 mM ammonium hydroxide, and finally washed twice in 50 mM ammonium hydroxide. Samples were loaded on the equilibrated StageTips and washed twice with 50 mM ammonium hydroxide. StageTip fractionation elution was performed with 40 μl of 50mM ammonium hydroxide containing increasing amounts of acetonitrile (5, 10, 15, 20, 27, 35%). Eluted sample fractions were dried to completion in a SpeedVac at 60°C, dissolved in 12 μl 0.1% formic acid, and stored at −20°C until MS analysis. For lysine acetylation analysis, mESCs were grown in conventional Serum/LIF media and switched to media with D-galactose (Sigma) replacing D-glucose and pyruvate (labeled Gal), with or without 20mM NAM (labeled NAM) and collected after 24 hrsh and compared to cells with a media change of conventional Serum/LIF (labeled Glu). Cells were washed three times in ice-cold PBS, before being harvested and resuspended with 10 volumes of 6M Guanidinium Hydrochloride (GndCl in 50mM Tris pH8.5) and snap frozen in liquid nitrogen. Pellets were then thawed, reduced and alkylated in 5mM TCEP and CAA at room temperature (RT), before sonication and measurement of protein content by Bradford (QIAGEN). Proteins were then digested with LysC (1:200) for 3h at RT, then dilution with 3 volumes of ammonium bicarbonate (ABC) and incubated with trypsin (1:200) overnight at RT. Peptides were then acidified to 0.5% TFA and loaded onto a SepPak C18 Classic Cartridge (VWR), eluted with 30% ACN in 0.1% TFA, frozen and lyophilized for 5 days. To enrich for acetylated peptides, samples were incubated with PTMScan Acetyl-Lysine (Ac-K) beads (CST) for 2h at 4°C, following the commercial protocol for buffer, wash and elution conditions. Peptides were then purified by StageTip (C18 material) and fractionated at high pH as described above. Specifically, samples were eluted with 75 μl of 2, 4, 7,10, 15, or 25% ACN in 50 mM ammonium hydroxide. Flow-through during sample loading was collected, acidified and reloaded onto a new StageTip that had been activated 30% CAN in 0.1% formic
P6826PC00 acid, washed two times with 0.1% formic acid. This fraction (F0) was then eluted with 75 uL of 25% ACN in 0.1% formic acid, then dried and reconstituted the same as the other fractions. Mass Spectrometry and Analysis: MS samples were analyzed on an EASY-nLC 1200 system (Thermo) coupled to either a Q Exactive HF-X Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo) for the proteome analysis or an Orbitrap Exploris 480 mass spectrometer (Thermo) for acetylome analysis. Separation of peptides was performed using 15-cm columns (75 μm internal diameter) packed in-house with ReproSil-Pur 120 C18-AQ 1.9 μm beads (Dr. Maisch). Elution of peptides from the column was achieved using a gradient ranging from buffer A (0.1% formic acid) to buffer B (80% acetonitrile in 0.1% formic acid), at a flow rate of 250 nl/min. Gradient length was 80 min per sample (proteome) and 70 min per sample (acetylome), including ramp-up and wash-out. The column was heated to 40° C using a column oven, and ionization was achieved using either a NanoSpray Flex ion source (Thermo). Spray voltage set at 2 kV, ion transfer tube temperature to 275 °C, and RF funnel level to 40%. Measurements were performed with a full scan range of 300-1,750 m/z, MS1 resolution of 60,000 (HF-X) or 120,000 (Exploris), MS1 AGC target of 3,000,000 (HF-X) or normalized AGC target of 200% (Exploris) and MS1 maximum injection time of 60 ms (HF-X) or auto (Exploris). Precursors with charges 2-6 were selected for fragmentation using an isolation width of 1.3 m/z and fragmented using higher-energy collision disassociation (HCD) with a normalized collision energy of 25%. Precursors were excluded from re-sequencing by setting a dynamic exclusion or 60 s. MS2 AGC target was set to 200,000 and minimum MS2 AGC target to 20,000 (HF-X) or normalized AGC target of 200% (Exploris). For proteome runs, MS2 maximum injection time was 60 ms, MS2 resolution was 30,000, and loop count was 12. For acetylome runs, a MS2 maximum injection time of auto, MS2 resolution of 60,000, and loop count was 7. Data Analysis: All MS RAW data were analyzed using the freely available MaxQuant software (Cox et al.2008), v.1.6.0.1 (proteome) and v.1.6.15 (acetylome) using the Andromeda search engine, and subsequent analysis was performed using Perseus v. 1.6.0.2 (proteome) or v.1.6.14 (acetylome). For generation of theoretical spectral libraries, the Mus musculus FASTA database was downloaded from UniProt on the 25th of July 2017 (proteome) or the 21st of July 2020 (acetylome). In silico digestion of
P6826PC00 proteins to generate theoretical peptides was performed with trypsin, allowing up to 3 missed cleavages. Allowed variable modifications were oxidation of methionine (default), protein N-terminal acetylation (default) for all samples. Additional variable modifications included Acetyl (K) and Phospho (STY). Second peptide search was enabled. Matching between runs was enabled with an alignment window of 20 min and a match time window of 0.7 min. For proteome, Label-free quantification (LFQ) was enabled based on Cox et al.2008 and maximum variable modifications per peptide were reduced to 3. Stringent MaxQuant 1% FDR data filtering at the PSM- and protein- levels was applied (default). For acetylome analysis, peptide intensities were median normalized in Perseus. Proteins or acetylated peptides only identified by one site and potential contaminants were removed and then data was filtered for proteins or sites identified in at least 75% of the replicates in at least one condition. Conditions were compared using a two-way ANOVA (proteome) or two sample Student’s T-tests (acetylome) with 5% FDR. Cytoscape software was used with the String App and Omics Visualizer to create protein networks with functional enrichment of GO Terms and KEGG pathways. Results: The inventors performed a global acetylome on ESCs cultured in Serum/LIF, EMM or EMM+NAM for 24h, before immunopurification of acetyl lysine, followed by quantitative MS. As expected, the number of acetylated regions was higher in EMM+NAM compared to Serum/LIF and EMM, since NAM is a potent inhibitor of Sirtuin deacetylases (Fig.5a, b, c). The inventors then identified the sites that were specifically deacetylated in EMM relative to the other two conditions in response to this fundamental shift in metabolism (Fig.5d, e). In EMM there were 1871 sites that were deacetylated, out of a total number of 6733 identified sites (data not shown). Moreover, the majority of these proteins were enriched in the nucleus (77%), with 12% enriched in chromatin, indicating that the major response to the acetylome occurs in the nucleus and therefore affects nuclear processes such as transcription (Fig.5f). The inventors identified acetylation sites associated with canonical pluripotency and ICM-related TFs that were Sirtuin-, but not EP300/CBP-regulated (e.g., SOX2, OCT4 (also known as Pou5f1), NANOG, TEAD4, YAP1, TFAP2C, MYC). Together with the previous observation that histones are generally deacetylated in EMM-cultured ESCs (Fig.4i), the findings suggest that these TFs could be responsible for Sirtuin-dependent enhancer regulation in response to metabolic change.
P6826PC00 To test whether EMM-induced deacetylation modulates enhancer-binding behavior for these factors, the inventors performed genome-wide CUT&Tag on the TFs SOX2, TEAD1 and KLF4, that were identified in the combined acetylome and IMAGE analysis. SOX2 binding to both open and closed enhancer regions was 3-4-fold stimulated by EMM (Fig.5i). While the binding of both TEAD1 and KLF4 is globally reduced in EMM (Fig.5j), their binding is enhanced at regions where SOX2 is bound, particularly in opening regions (Fig.5k-m), showing that TF cooperativity at active enhancers is induced by Sirt1-induced deacetylation in EMM. To further show this, the authors generated an ESC line in which SIRT1 could be rapidly degraded with the addition of a compound called dTag. The occupation of SOX2 at opening enhancers, which is increased in EMM, was rescued by removing SIRT1 with dTag similar to the addition of NAM (Fig.5n). Conclusions. These data show that SIRT1 is required for increased SOX2 binding at enhancers in EMM, as well as for the deacetylation of histones at regions not required for the generation of an ICM phenotype (see model, Fig.5o). Consistent with the overall reduction in transcription levels the inventors observed a reduction in the number of active enhancers, from 13705 to 3829 (Fig.5h), but at enhancers where the chromatin opened, the inventors observed an increased level of acetylation, suggesting higher levels of activity (Fig.5g); a trend reflected in the opening of the shared enhancer set over time in EMM (data not shown). Conversely, the inventors also noted a reduction of acetylation at enhancers where the chromatin is closing (Fig.5g). Example 6. Expression of embryonic and extraembryonic markers in RSet hPSC in N2B27 or EMM Methods: Culture of naive hPSC in standard RSet -/+ EMM media. H9 hPSCs were cultured in chemically defined RSet media (Stem Cell Technologies), as previously described in Gafni et al., 2013. Cell were cultured in a humidified incubator in 5% oxygen, and were passaged as single cells every 3-4 days. hESC were also cultured in N2B27+t2iLGo (naïve) and DMEM-F12 (Gibco) +KnockOUT Serum Replacement (Thermo Fisher), MEM Non-Essential Amino Acids (Thermo Fisher), B- mercaptoethanol (Thermo Fisher) and 10ng/ml FGF2 (Peprotech) (primed), as control conditions.
P6826PC00 Real-Time qPCR Total RNA was collected using either Trizol (Invitrogen) or the RNeasy Mini Kit (QIAGEN). Genomic DNA was eliminated by DNase treatment (QIAGEN), and 1 μg of total RNA was used for first-strand synthesis with SuperScript III reverse transcriptase according to the manufacturer’s instructions. cDNA corresponding to 10ng total RNA was used for real-time (RT)-qPCR analysis using the Roche LC480, and target amplification was detected with the Universal Probe Library system. Values were normalized to the geometric mean for GAPDH and ACTB expression. Results: The inventors analysed the relative expression levels of RNA transcripts for both epiblast (NANOG, OCT4) and extra-embryonic hypoblast (PDGFRA) markers in hESCs cultured in RSet media with or without 4.5g/L D-Galctose supplementation (RSet and RSet+EMM, respectively). hESCs cultured in N2B27+t2iLGo (naïve) and DMEM-F12 (Gibco) +KnockOUT Serum Replacement (primed) were used as controls. The investigators found that while expression levels of epiblast markers NANOG and OCT4 did not differ significantly between RSet -/+ EMM (Fig.6a-b), they found that expression of the hypoblast marker PDGFRA was higher in RSeT+EMM, similar to levels in “primed” hESC (Fig.6c). Conclusion: The investigators conclude that upregulating OXPHOS and downregulating glycolysis in RSet-cultured hESC may increase levels of extra-embryonic gene expression in hESC, while the levels of epiblast markers appear relatively unchanged, similar to the effect in mESCs (see Example 1). Example 7. Creation of blastoids in EMM Methods Culture of naive hPSC in standard N2B27 or EMM media. H9 hPSCs were cultured in chemically defined N2B27 media, composed of DMEM/F12 (Gibco), Neurobasal media (Gibco), N2 (made in house) and B27 (Gibco). The standard culture media consisted of N2B27 was supplemented with 10ng/ml recombinant human LIF (Peprotech), 3 µM Gö6983 (Tocris), 1 µM PD0325901 (Tocris) and 1µM CHIR99021 (Sigma-Aldrich). Variations of this culture media were: N2B27 composed of glucose-free DMEM-F12 and Neurobasal media (both Gibco),
P6826PC00 supplemented with PXGL (the same combination of inhibitors and cytokines as described above, except with 3uM XAV939 (Tocris) replacing CHIR99021), and 21.25mM D-Glucose with 0.365mM Sodium Pyruvate, or 21.25mM D-Galactose (All Thermo Fisher). hESCs were also cultured in DMEM with glucose and pyruvate or DMEM with galactose (see Methods in Example 1). hESCs were cultured in 5% or 20% oxygen conditions, and passaged as single cells every 3-4 days. Blastoid formation hESCs were treated with Accutase (Biozym) at 37 °C for 5 min, followed by gentle dissociation with a pipette. After centrifugation, the cell pellet was resuspended in PXGL medium, supplemented with 10 μM Y-27632 (MedChemExpress). Cell number was determined using a Countess automated cell counter (Thermo Fisher Scientific). The cells were then resuspended in N2B27 medium containing 10 μM Y-27632 (aggregation medium) and 1.0 × 105 cells were seeded onto a microwell array included into a well of a 96-well plate and placed in a hypoxic chamber (5% CO2 , 5% O2 ) for the whole period of blastoid formation. The cells were allowed to form aggregates inside the microwell for a period ranging from 3H, then the aggregation medium was replaced with PALLY medium (N2B27 supplemented with 1 μM PD0325901, 1 μM A 83-01 (MedChemExpress), 500 nM 1-oleoyl lysophosphatidic acid sodium salt (LPA,(Tocris), 10ng/ml hLIF and 10 μM Y-27632. The PALLY medium was refreshed every 24 h. After 48 h, the PALLY medium was replaced with N2B27 medium containing 500 nM LPA and 10 μM Y-27632. At 96 h, the blastoids were collected for further analysis. Analysis of Lineage specific markers in blastoids by immunofluorescence Blastoids were washed and fixed in 4% formaldehyde (Fisher Scientific, PI-28906), blocked, and permeabilized in 5% donkey serum and 0.3% Triton. Antibodies were incubated overnight in 1% BSA and 0.3% Triton in PBS and subsequently visualized with the appropriate secondary antibody (Alexa Fluor, Molecular Probes) and imaged using a Leica TCS SP8 confocal microscope and analyzed using Imarisx649.5.1 software. Results The investigators first attempted to culture H9 hESC monolayer culture in various culture media containing different combinations of base media, inhibitors, cytokines
P6826PC00 and metabolic components, and assess the phenotype of the cells after 48h (Fig.7). The control media of N2B27+t2iLGo produced hESC that have the classic “naïve” phenotype, with cells packed into tight domes. hESC cultured in DMEM also produced naïve cell colonies when supplemented with glucose and pyruvate, but when supplemented with galactose, there was a higher level of cell death, and fewer naïve cell colonies. hESC were also cultured in N2B27+PXGL -/+EMM, and interestingly both conditions generated hESC colonies that appeared naïve. However, when cultured at 20% oxygen the level of cell death increased, as did the proportion of differentiated cells. Next, the investigators attempted to develop blastoids from H9 hESC, in the presence or absence of EMM (also -/+ insulin) (Fig.8).EMM culture was able to generate phenotypically normal blastoids, although they appeared slightly smaller compared to the control blastoids, To assess the lineage contribution of the EMM-cultured blastoids, the investigators then performed immunofluorescence staining on markers for the three lineages: epiblast (KLF17), hypoblast (GATA4) and trophoblast (GATA3) (Fig.9a). They found that when they quantified the numbers of blastoids containing GATA4+ cells, the blastoids cultured in EMM-/+insulin had a higher proportion of GATA4+ blastoids, when compared to the control conditions (Fig.9b). Conclusion In this example, the investigators show that there are conditions in which naïve hESC can be successfully cultured in EMM conditions, and that blastoids can be created in these conditions as well. The investigators also demonstrate that the upregulation of OXPHOS and downregulation of glycolysis during blastoid formation increases the proportion of blastoids with hypoblast (GATA4+) cells. References Ryall, J. G. et al. The NAD+-dependent sirt1 deacetylase translates a metabolic switch into regulatory epigenetics in skeletal muscle stem cells. Cell Stem Cell 16, 171–183 (2015). Canham, M. A., Sharov, A. A., Ko, M. S. H. & Brickman, J. M. Functional heterogeneity of embryonic stem cells revealed through translational amplification of an early endodermal transcript. PLoS Biol.8, (2010).
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