Patent Application
20240018470
The ASCII text file named “047162-7368US1_SeqListing.xml” created Jul. 11, 2023, comprising 17.4 Kbytes, is hereby incorporated by reference in its entirety.
During certain stages of the embryonic development, cells need to extinguish pluripotency factors so as to activate pathways of cellular differentiation. The understanding of the factors controlling this step would allow the manipulation of pluripotency in cells, for example the ability to maintain pluripotency in stem cells. Therefore, there is a need to elucidate factors that control the exit of cells from pluripotency. This knowledge would allow for rationally manipulating pluripotency in cells. The present invention addresses this need.
In some aspects, the present invention is directed to the following non-limiting embodiments.
In some embodiments, the present invention is directed to a method of maintaining pluripotency in a cell.
In some embodiments, the method comprises at least one of the following: reducing the membrane potential of the cell; activating a voltage gated calcium channel on the plasma membrane of the cell; and/or increasing the calcium ion concentration in the cell.
In some embodiments, reducing the membrane potential of the cell comprises at least one of the following: subjecting the cell to an extracellular environment having a high concentration of potassium ions; inhibiting a potassium channel on the plasma membrane of the cell; and/or contacting the cell with a potassium selective ionophore.
In some embodiments, reducing the membrane potential of the cell comprises subjecting the cell to an extracellular environment having a potassium ion concentration of about 0.5 mM or higher, such as about 0.75 mM or higher, about 1 mM or higher, about 2 mM or higher, about 3 mM or higher, about 4 mM or higher, about 5 mM or higher, about 7.5 mM or higher, about 10 mM or higher, about 12.5 mM or higher, about 15 mM or higher, about 20 mM or higher, about 25 mM or higher, about 30 mM or higher, about 40 mM or higher, or about 50 mM or higher.
In some embodiments, reducing the membrane potential of the cell comprises inhibiting a potassium channel on the plasma membrane of the cell, wherein the potassium channel comprises an inwardly-rectifying voltage gated potassium channel.
In some embodiments, the inwardly-rectifying voltage gated potassium channel comprises potassium voltage-gated channel subfamily H member 6 (KCNH6).
In some embodiments, inhibiting the potassium channel on the plasma membrane of the cell comprises contacting the cell with a potassium channel inhibitor.
In some embodiments, the inhibitor of the potassium channel comprises barium ions or an Ergtoxin.
In some embodiments, reducing the membrane potential of the cell comprises contacting the cell with a potassium selective ionophore comprising valinomycin, BME 44 (2-Dodecyl-2-methyl-1,3-propanediyl bis[N-[5′-nitro(benzo-15-crown-5)-4′-yl]carbamate]), or BB15C5 (Bis[(benzo-15-crown-5)-4′-ylmethyl] pimelate).
In some embodiments, the voltage gated calcium channel is an L-type calcium channel or a T-type calcium channel.
In some embodiments, the cell is a stem cell.
In some embodiments, the cell is an embryonic stem cell.
In some embodiments, the cell is in an organism, a cultured primary cell, or a cultured cell line.
In some embodiments, the cell is from a vertebrate origin.
In some embodiments, the cell is from a mammalian origin.
In some embodiments, the cell is from a human origin.
In some aspects, the present invention is directed to a composition.
In some embodiments, the composition comprises a culture medium and a pluripotent cell.
In some embodiments, potassium ions in the culture medium are present in a concentration of about 0.5 mM or higher.
In some embodiments, a pluripotency of the pluripotent cell is maintained by the concentration of potassium ions in the culture medium.
In some embodiments, the concentration of the potassium ions in the culture medium reduces the membrane potential of the cell, thereby maintaining the pluripotency of the pluripotent cell.
In some embodiments, the culture medium comprises: at least one inorganic ion selected from a sodium ion, a potassium ion, a calcium ion, a magnesium ion, a chloride ion, a sulfate ion, a carbonate ion, a bicarbonate ion, a phosphate ion, a phosphate monobasic ion, a phosphate dibasic ion; an amino acid; and a vitamin.
In some embodiments, the amino acid comprises at least one selected form the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
In some embodiments, the vitamin comprises at least one selected from the group consisting of pantothenate, choline, folic acid, inositol, nicotinamide, pyridoxine, riboflavin, and thiamine.
In some embodiments, the culture medium further comprises a carbohydrate.
In some embodiments, the culture medium further comprises one or more selected form the group consisting of pyruvate, lipoic acid, biotin, a buffering agent, and a pH indicator.
In some embodiments, the pluripotent cell is a stem cell or an embryonic stem cell.
In some embodiments, the pluripotent cell is from a vertebrate origin.
In some embodiments, the pluripotent cell is from a mammalian origin.
In some embodiments, the pluripotent cell is from a human origin.
In some embodiments, the pluripotent cell does not have the cell potency to develop in to a human.
The following detailed description of exemplary embodiments will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating, non-limiting embodiments are shown in the drawings. It should be understood, however, that the instant specification is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.
The following disclosure provides many different embodiments, or examples, for implementing different features of the provided subject matter. Specific examples of components and arrangements are described below to simplify the present disclosure. These are, of course, merely examples and are not intended to be limiting. For example, the formation of a first feature over or on a second feature in the description that follows may include embodiments in which the first and second features are formed in direct contact, and may also include embodiments in which additional features may be formed between the first and second features, such that the first and second features may not be in direct contact. In addition, the present disclosure may repeat reference numerals and/or letters in the various examples. This repetition is for the purpose of simplicity and clarity and does not in itself dictate a relationship between the various embodiments and/or configurations discussed.
The study described herein (“the present study”), using xenopus embryos (such as embryos at blastula or gastrula stages) and human embryonic stem cells (hESCs) as non-limiting examples, discovered that pluripotency of cells, such as embryonic cells or stem cells, can be maintained by reducing the cellular membrane potentials (i.e., depolarizing the cells).
Accordingly, in some aspects, the present invention is directed to a method of maintaining pluripotency in a cell.
The present study further discovered that, one way to depolarize cell membranes and maintain pluripotency in the cell is to culture the cell in a medium having higher than usual potassium ion concentrations.
Accordingly, in some aspects, the present invention is directed to a culture medium for maintaining pluripotency in cultured cells.
As used herein, each of the following terms has the meaning associated with it in this section. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Generally, the nomenclature used herein and the laboratory procedures in animal pharmacology, pharmaceutical science, peptide chemistry, and organic chemistry are those well-known and commonly employed in the art. It should be understood that the order of steps or order for performing certain actions is immaterial, so long as the present teachings remain operable. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting; information that is relevant to a section heading may occur within or outside of that particular section. All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference.
In the application, where an element or component is said to be included in and/or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components and can be selected from a group consisting of two or more of the recited elements or components.
In the methods described herein, the acts can be carried out in any order, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.
In this document, the terms “a,” “an,” or “the” are used to include one or more than one unless the context clearly dictates otherwise. The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. The statement “at least one of A and B” or “at least one of A or B” has the same meaning as “A, B, or A and B.”
“About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, in certain embodiments ±5%, in certain embodiments ±1%, in certain embodiments ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
The present study, using xenopus embryos (such as embryos at blastula or gastrula stages) and human embryonic stem cells (hESCs) as non-limiting examples, discovered that pluripotency of cells, such as embryonic cells or stem cells, can be maintained by reducing the membrane potential of the cells.
Accordingly, in some aspects, the present specification is directed to a method of maintaining pluripotency in a cell.
In some embodiments, the method includes reducing the cell membrane potential; activating a voltage gated calcium channel in the cell membrane (plasma membrane); and/or increasing calcium ion concentration in the cell.
As used herein, the term “reducing membrane potential” means bringing the membrane potential of the cell closer to 0. For example, if a membrane potential of the cell is about −70 mV, the membrane potential is considered to be reduced if the membrane potential is brought to about −60 mV, −50 mV, −40 mV, −30 mV, and/or −20 mV. The present specification sometimes refers to “reducing membrane potential” of a cell as “depolarizing” the membrane of the cell.
In some embodiments, reducing the membrane potential of the cell includes: subjecting the cell to an extracellular environment having a high concentration of potassium ions; inhibiting a potassium channel in a plasma membrane of the cell; or contacting the cell with a potassium selective ionophore.
In some embodiments, subjecting the cell to an extracellular environment having a high concentration of potassium ions includes subjecting the cell to an extracellular environment having a concentration of potassium ions of about 0.5 mM or higher, such as about 0.75 mM or higher, about 1 mM or higher, about 2 mM or higher, about 3 mM or higher, about 4 mM or higher, about 5 mM or higher, about 7.5 mM or higher, about 10 mM or higher, about 12.5 mM or higher, about 15 mM or higher, about 20 mM or higher, about 25 mM or higher, about 30 mM or higher, about 40 mM or higher, or about 50 mM or higher. In some embodiments, subjecting the cell to an extracellular environment having a high concentration of potassium ions includes culturing the cell in a culture medium having an alleviated potassium ion concentration, such as the concentrations described herein.
In some embodiments, reducing the membrane potential of the cell includes inhibiting the potassium channel on the plasma membrane of the cell. In some embodiments, the potassium channel comprises an inwardly-rectifying voltage gated potassium channel. In some embodiments, the inwardly-rectifying voltage gated potassium channel includes potassium voltage-gated channel subfamily H member 6 (KCNH6).
In some embodiments, inhibiting the potassium channel on the plasma membrane of the cell comprises contacting the cell with an inhibitor of the potassium channel. In some embodiments, the inhibitor of the potassium channel includes barium ions or an Ergtoxin.
In some embodiments, reducing the membrane potential of the cell comprises contacting the cell with a potassium selective ionophore. In some embodiments, the potassium selective ionophore comprises valinomycin, BME 44 (2-Dodecyl-2-methyl-1,3-propanediyl bis[N-[5′-nitro(benzo-15-crown-5)-4′-yl]carbamate]), or BB15C5 (Bis[(benzo-15-crown-5)-4′-ylmethyl] pimelate).
In some embodiments, the calcium channel is an L-type calcium channel or a T-type calcium channel.
In some embodiments, the cell which pluripotency is maintained by the method is a stem cell. In some embodiments, the cell is an embryonic stem cell. In some embodiment the cell is in an organism, a cultured primary cell, or a cultured cell line. In some embodiments, the cell is from a vertebrate origin, such as from a mammalian origin, or from a human origin.
The present study discovered that one way to depolarize cell membranes and maintain pluripotency in the cell is to culture the cell in a culture medium having higher than usual potassium ion concentrations.
Accordingly, in some aspects, the present specification is directed to a culture medium. In some embodiments, the culture medium is for maintaining a pluripotency of a cell cultured therein.
In some embodiments, the culture medium includes at least one of the following:
In some embodiments, the concentration of potassium ions in the culture medium is about 0.5 mM or higher, such as about 0.75 mM or higher, about 1 mM or higher, about 2 mM or higher, about 3 mM or higher, about 4 mM or higher, about 5 mM or higher, about 7.5 mM or higher, about 10 mM or higher, about 12.5 mM or higher, about 15 mM or higher, about 20 mM or higher, about 25 mM or higher, about 30 mM or higher, about 40 mM or higher, or about 50 mM or higher.
In some embodiments, the one or more amino acids include at least one of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, or combinations thereof. In some embodiments, some of the amino acids are L-amino acids. In some embodiments, all of the amino acids are L-amino acids.
In some embodiments, the one or more vitamins include pantothenate, choline, folic acid, inositol, nicotinamide, pyridoxine, riboflavin, thiamine, or combinations thereof.
In some embodiments, the culture medium further includes one or more carbohydrates. In some embodiments, the one or more carbohydrates include D-glucose.
In some embodiments, the culture medium further includes one or more of pyruvate, lipoic acid, biotin, a buffering agent (such as HEPES), a pH indicator (such as phenol red).
In some embodiments, the culture medium is the same as or similar to existing culture media, such as RPMI 1640 (Moore et al, JAMA. 1967 Feb. 20; 199(8):519-24.), Iscove's Modified Dulbecco's Medium (IMDM), Minimum Essential Medium Eagle (MEM) (Eagle, Science. 1959 Aug. 21; 130(3373):432-7) or Dulbecco's Modified Eagle Medium (DMEM) (Dulbecco et al., Virology, Volume 8, Issue 3, July 1959, Pages 396-397), except for the alleviated concentrations of potassium ions. In some embodiments, the concentration of each of the non-potassium components is within about ±20%, such as about ±15%, about ±12.5%, about ±10%, about ±7.5%, about ±5%, about ±2% or about ±1% of the currently used concentrations.
In some embodiments, the cell which pluripotency is maintained by the culture media is a stem cell. In some embodiments, the cell is an embryonic stem cell. In some embodiment the cell is in an organism, a cultured primary cell, or a cultured cell line. In some embodiments, the cell is from a vertebrate origin, such as from a mammalian origin, or from a human origin.
The present study discovered that keeping pluripotent cells in in a culture medium having higher than usual potassium ion concentrations is able to maintain the pluripotency in the cell.
Accordingly, in some aspects, the present invention is directed to a composition including a pluripotent cell. In some embodiments, other components in the composition maintains the pluripotency of the pluripotent cell.
In some embodiments, the composition includes a pluripotent cell and a culture medium. In some embodiments, the culture medium is the same as or similar to those as described elsewhere herein, such as in the “Culture Medium for Maintaining Pluripotency” section.
In some embodiments, the pluripotent cell is a stem cell. In some embodiments, the pluripotent cell is an embryonic stem cell. In some embodiment the pluripotent cell is in an organism, a cultured primary cell, or a cultured cell line. In some embodiments, the pluripotent cell is from a vertebrate origin, such as from a mammalian origin, or from a human origin. In some embodiments, the pluripotent cell does not have the cell potency to develop in to a human.
The present specification further describes in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless so specified. Thus, the present specification should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.
Transitioning from pluripotency to differentiated cell fates is fundamental to both embryonic development and adult tissue homeostasis. Improving the understanding of this transition would facilitate the ability to manipulate pluripotent cells into tissues for therapeutic use.
Referring to the Example 2 section herein, the study described herein (“the present study”) demonstrates that membrane voltage (Vm) regulates the exit from pluripotency and the onset of germ layer differentiation in the embryo, a process that affects both gastrulation and left-right patterning. By examining candidate genes of congenital heart disease and heterotaxy, KCNH6, a member of the ether-a-go-go class of potassium channels that hyperpolarizes the Vm and thus limits the activation of voltage gated calcium channels, lowering intracellular calcium, was identified. In pluripotent embryonic cells, depletion of kcnh6 led to membrane depolarization, elevated intracellular calcium levels, and the maintenance of a pluripotent state at the expense of differentiation into ectodermal and myogenic lineages. Using high-resolution temporal transcriptome analysis, the present study identifies the gene regulatory networks downstream of membrane depolarization and calcium signaling and discover that inhibition of the mTOR pathway transitions the pluripotent cell to a differentiated fate. By manipulating Vm using a suite of tools, the present study establishes a bioelectric pathway that regulates pluripotency in vertebrates, including human embryonic stem cells.
Transitioning from pluripotency to differentiated cell fates is fundamental to both embryonic development and adult tissue homeostasis. Improving the understanding of this transition would facilitate the ability to manipulate pluripotent cells into tissues for therapeutic use. Here, the present study shows that membrane voltage (Vm) regulates the exit from pluripotency and the onset of germ layer differentiation in the embryo, a process that affects both gastrulation and left-right patterning. By examining candidate genes of congenital heart disease and heterotaxy, the present study identified KCNH6, a member of the ether-a-go-go class of potassium channels that hyperpolarizes the Vm and thus limits the activation of voltage gated calcium channels, lowering intracellular calcium. In pluripotent embryonic cells, depletion of kcnh6 led to membrane depolarization, elevated intracellular calcium levels, and the maintenance of a pluripotent state at the expense of differentiation into ectodermal and myogenic lineages. Using high-resolution temporal transcriptome analysis, the present study identified the gene regulatory networks downstream of membrane depolarization and calcium signaling and discover that inhibition of the mTOR pathway transitions the pluripotent cell to a differentiated fate. By manipulating Vm using a suite of tools, the present study established a bioelectric pathway that regulates pluripotency in vertebrates, including human embryonic stem cells.
Action potentials are fundamental to the function of excitable cells, including neurons, cardiomyocytes and pancreatic cells. They are produced through tightly orchestrated changes in the membrane potential (Vm). However, most animal cells, excitable or not, have a resting state Vm (resting membrane potential) that depends on a) the permeability of the plasma membrane for each ion (p in the Goldman-Hodgkin-Katz (GHK) equation,
In order to respond, voltage sensitive effector molecules depend on the magnitude in the change of Vm. Quantitative Vm measurements in early embryos are rare but were performed in the 1960s and 1970s from 1 cell stage embryos through blastula stages in Triturus and Xenopus embryos. The blastula embryo has completed a series of rapid cell divisions (cleavages), has established germ layer cell fates (ectoderm, mesoderm, and endoderm), and is poised to begin gastrulation, the process by which cell movements transform the embryo to acquire the adult body plan. Notably, while the Vm at early cleavage stages is depolarized (=more positive Vm) (Vm@2-cell=−19±10 mV), it becomes progressively hyperpolarized (=more negative) towards blastula stages (−50 mV). The implications of this progressive Vm polarization during early development are unclear, as is a mechanism by which Vm could transduce a signal within embryonic cells or act complementary to signals transduced biochemically (i.e. ligand-receptor).
To address the question of when Vm is critical for embryonic development, the present study employed barium ions to block K+ channels at different time points of embryonic development, since K+ conductance is paramount for determining Vm. Because K+ conductance drives the membrane voltage to a negative (hyperpolarized) potential, blocking K+ channels depolarizes cells. In line with the electrophysiological evidence demonstrating that embryos first become polarized at the blastula stage, the present study found that Barium treatment affected embryonic development primarily when embryos were treated from blastula stages through gastrulation rather than at earlier cleavage stages (
Recent studies in patients with congenital heart disease identified a number of variants in KCNH ether-a-go-go (EAG) potassium channels (
In Xenopus, the present study found kcnh6 to be expressed in the prospective ectoderm and dorsal/paraxial mesoderm at gastrulation onset, suggesting that it could play a role during gastrulation (
Depletion/inhibition of potassium channels or elevation of extracellular K+ should lead to membrane depolarization. Thus, it was reasoned that the inverse condition, namely hyperpolarizing by reducing extracellular K+, should rescue kcnh6 depleted embryos. Lowering extracellular K+ ([K]o in GHK eq.
Finally, the present study sought to measure the change in Vm when kcnh6 is depleted. Using intracellular electrodes in the animal pole of kcnh6 MO vs control MO injected embryos at gastrulation onset, the present study recorded a Vm of −21.8±4.6 mV in kcnh6 MO vs −44±6.8 mV in control MO embryos (
The present study then asked how Vm is transduced into a signal that affects embryonic development. There are a limited number of voltage responsive elements in a cell. It was reasoned that depolarization (Vm=−20 mV) in kcnh6 depleted embryos could aberrantly activate voltage-gated Ca′ channels (VGCCs), which facilitate inward Ca′ flux. L-type VGCCs are present in the prospective ectoderm and dorsal mesoderm and can induce potent intracellular Ca′ increases that can alter germ layer patterning, yet upstream regulators of these calcium channels remain elusive. Interestingly, intracellular Ca′ is elevated after fertilization and during early cleavage stages but declines as the embryo approaches gastrulation 25 concomitant with the onset of membrane polarization. It was argued that, if VGCCs are aberrantly activated due to an abnormally depolarized Vm, the present study should be able to detect changes in intracellular Ca′ levels. To assess this, the present study microinjected the calcium indicator GCaMP626 mRNA together with mCherry mRNA (to enable ratiometric analysis) into control MO or kcnh6 MO embryos and performed calcium imaging in animal cells of early gastrula embryos. Within the animal pole of stage 10 control MO-injected embryos, the present study observed multiple intracellular calcium increases, signified by a pulse-like appearance of GCaMP6 fluorescence in isolated cells, which then propagated to adjacent cells. These increases are well documented in Xenopus stage 8 to 12 gastrulae, i.e. last a few seconds, in which they spread to adjacent cells and then extinguish, are VGCC dependent and may contribute to neural induction. The present study confirmed the existence of Ca2+ transients at stage 10 by performing 20s time lapse recordings, and additionally observed that they are of low intensity and typically do not simultaneously affect more than 16±10% of the total animal pole area (
For gastrulation to proceed normally, two steps are critical: first, the germ layers of the blastula embryo (ectoderm, mesoderm, and endoderm) must be patterned correctly and second, the embryo must undergo the cellular rearrangements that drive morphogenesis. Calcium plays a role in morphogenesis cell behaviors during gastrulation. Alternatively, calcium may play a role in patterning the mesoderm that also drives gastrulation cell movements. Patterning precedes morphogenesis, and morphogenesis can fail as a result of abnormal patterning. We, therefore, first examined if patterning is disrupted in Vm-depolarized embryos via marker gene expression. Since the mesoderm is critical for gastrulation movements, the present study began with this germ layer. Markers of the dorsal (gsc, nodal3) and ventral mesoderm (vent2) appeared unaffected in kcnh6 depleted, barium and high K+ depolarized embryos (
When certain biochemical signaling factors are depleted, loss of one cell fate (e.g. paraxial mesoderm) is often concomitant with gain of another cell fate. Since the dorsal or ventral mesoderm appeared unaffected (
In depolarized embryos, the present study has established 1) changes in cell fate and 2) elevated intracellular calcium levels, so the present study next tested if these aberrant cell fates are dependent on voltage gated calcium channels. To determine the specific embryonic VGCCs downstream of Vm, the present study reviewed the available high temporal resolution RNA-Seq data. Xenopus contains detectable transcripts of L- and T-type VGCCs between the 1-cell and gastrula stages, while other VGCC types (N-, R- and P/Q) are less abundant. L-type VGCCs become activated at Vm>−40 mV (and then inactivated at Vm>10 mV) and are implicated in gastrula patterning while T-type channels become inactivated at Vm>−60 mV and would be inactive both at physiological Vm (˜−50 mV) and at more depolarized potentials. Therefore, the present study tested the L-type VGCC blocker nifedipine. This significantly ameliorated both ectodermin (ectoderm) and myf5 (paraxial mesoderm) expression losses in kcn6 knockdown embryos (
The loss of some cell fates (ectoderm and paraxial mesoderm) without a concomitant expansion of other cell fates was puzzling given that most biochemical signaling factors (Wnt, BMP, Nodal) generally balance different cell fates in the early embryo. It was speculated that these unspecified cells may simply lack the ability to assume any cell fate because they remain pluripotent abnormally. To test this hypothesis, the present study examined markers of pluripotency OCT4, NANOG, and SOX2. In Xenopus, there are three OCT4 homologs (pou5f3.1, 2 and 3, formerly oct91, oct25 and oct60), and the ventx1.2/2.2 factors, which have overlapping functions in maintaining differentiation competence and are thought to be structurally and functionally equivalent to mammalian Nanog40. Sox2, a core pluripotency factor in mammals, is highly conserved in amphibians and also expressed at high levels prior to lineage commitment throughout the Xenopus blastula41,42. The present study examined the prospective ectoderm of embryos, which is best characterized in its pluripotent properties and confirmed that pou5f3.1, pou5f3.3, sox2 and ventx1.2 are robustly expressed at stage 9 prior to lineage commitment, but their transcripts are sharply reduced by stage 10 in control embryos (
Based on this result, the present study sought to test the pluripotency of these kcnh6-depleted embryos. In the blastula (stage 9), the prospective ectoderm or “animal cap” contains cells that when explanted will differentiate into epidermis (
Our findings indicate that a polarized Vm limits voltage-gated calcium channels and intracellular calcium, a process that reduces the expression of pluripotency genes as germ layer differentiation initiates. A critical question is what are the signaling pathways invoked when Vm is depolarized or intracellular calcium is elevated. To address this question in an unbiased manner, the present study temporally profiled gene expression via RNA-Seq in control and high K+ depolarized embryos by collecting embryos every 30 min from pre- to post-gastrula stages (stages 8 to 12;
The present study found a hierarchy of gene set enrichments from early to late in the time course, reflecting the changing response in the transcriptome (
To build the underlying gene regulatory networks, the present study examined transcription factor motif enrichment in the promoters of each of these activated gene clusters. Mirroring the gene set enrichments, the present study found motif enrichments segregated between early (A1,2) and later (A3,4) gene clusters (
Turning to the genes activated later, particularly, those associated with pluripotency and germ layer commitment in cluster A4, the present study found comprehensive enrichment of FOXH1, SOX and POU motifs in their promoters (
The transcriptome analysis herein not only revealed a potential gene regulatory network but pointed towards a role for mTOR. mTOR is critical for multiple cellular processes including autophagy, nutrient sensing, and an emerging role in pluripotency. Because the expression of mTOR pathway members was increased in depolarizing conditions and pathways associated with mTOR, it was reasoned that mTOR signaling was upregulated and maintained pluripotency in these depolarized embryos. To test this hypothesis, the present study applied the mTORC1 inhibitor, rapamycin, to depolarized gastrulating embryos to see if this could abolish the aberrant expression of pluripotency markers pou5f3.3 and ventx1.2 in the animal pole and activate germ layer differentiation. Rapamycin dramatically lowered expression of pou5f3.3 and ventx1.2 in the animal pole in kcnh6 CR and high K+ treated embryos compared to those embryos treated with vehicle alone and appeared comparable to untreated control embryos (
Finally, the present study tested whether the findings herein would also apply to human embryonic stem cells (hESCs). At stage 9, Xenopus animal cap cells are pluripotent in that they can, under appropriate conditions, form derivatives of any of the three germ layers (
The present study also tested whether blocking K+ channels with Ergtoxin affected the kinetics of differentiation. BMP4 induces differentiation to either mesodermal or extraembryonic fates in a dose-dependent manner. Ergtoxin caused a significant delay in downregulation of pluripotency markers such as SOX2 at 12 hours with a similar trend in NANOG (
To test whether the role of mTOR signaling downstream of membrane depolarization is conserved, the present study treated hESCs with rapamycin with or without Ergtoxin. Treatment with rapamycin led to reduction of pluripotency markers in a dose dependent manner with near complete loss by 5 days (
A model in which membrane voltage regulates intracellular calcium during a critical stage of embryonic development, at which point cells need to extinguish pluripotency factors in order to activate a program of cellular differentiation, is proposed herein (
The present study showed that regulation of voltage gated calcium channels by Vm is critical for the exit from pluripotency. Based on this, it is suggested that low intracellular calcium reduces the expression of mTOR and pluripotency factors, which is conducive to differentiation onset.
The work herein connects Vm and intracellular calcium as upstream of this pluripotency program. The work herein demonstrates the importance of Vm in vivo during early embryonic development as well as in vitro in human stem cells. Importantly, this pathway is readily manipulated by a wide range of highly specific channel inhibitors or simple changes in extracellular ionic concentrations. Therefore, the present study defines multiple tools for pluripotency manipulations in embryos, organoids, and adult tissues where stem cells play a critical role.
Adult Xenopus tropicalis were raised and housed according to the established protocols which were approved by the Yale Institutional Animal Care and Use Committee. The present study induced ovulation, performed IVF, and raised embryos in 1/9×MR. The present study staged X. tropicalis embryos according to Nieuwkoop and Faber (Normal table of Xenopus laevis (Daudin): a systematical and chronological survey of the development from the fertilized egg till the end of metamorphosis. (Garland Pub., 1994))
Morpholino Oligonucleotides, mRNA and CRISPRs
All injections of Xenopus embryos were performed at the one-cell stage using a fine glass needle and Picospritzer system. A kcnh6 translation blocking (kcnh6 MO, 5′-GGTCCTCGAAGTTTAGGATAAACAT-3′, SEQ ID NO:1) and a scrambled morpholino oligonucleotide were obtained from Gene Tools LLC and injected at 10 ng to deplete kcnh6 or as a control, respectively. CRISPR sgRNAs for kcnh6 targeted either exon 3 or exon 4 based on the v7.1 gene model of the X. tropicalis genome (CRex3: 5′-GGAATAAGGGGTGAAGACAGCGG-3′, SEQ ID NO:2 and CRex4: 5′-AGGGCGCTCTACATTTCCAATGG-3′, SEQ ID NO:3). CRISPR sgRNAs for cacna1c (5′-GCAGACGGGGGCAGCGCCATTGG-3′, SEQ ID NO:4) and cacna1g (5′-GGTTAATGGCTCTCAGCGGGCGG-3′, SEQ ID NO:5) were designed from the v7.1 model of the Xenopus tropicalis genome. For F0 CRISPR knockdown, embryos were injected with 1.5 ng Cas9 Protein (PNA-Bio) and 400 pg of targeting sgRNA and raised to desired stages. For pitx2 and coco expression analyses, the dose of kcnh6 sgRNA was halved to a subphenotypic dose of 200 pg to obtain embryos without gross morphological gastrulation defects. Full length human KCNH6 (NM 030779.3; cloned in pCS107), GCaMP6 (subcloned in pCSDest) and mCherry cDNAs (Addgene #34935; in pCS2+), were used to generate capped mRNAs in vitro by first linearizing with appropriate restriction enzymes and then transcribing with the mMessage machine kit (Ambion). mRNAs were injected at 3 pg (human KCNH6), 150 pg (GCaMP6) and 150 pg (mCherry) per embryo. Embryos were raised at 21° C. to allow time for sufficient expression levels at blastula/gastrula stages.
Genomic DNA from CRISPR and control embryos were obtained by lysing individual, stage 45 tadpoles in 50 mM NaOH and amplifying PCR fragments around the CRISPR target site that encompass approximately 200 bp upstream and 500 bp downstream of the site. The following primers were used for CRISPRs targeting exons 3 and 4 of the kcnh6 locus, respectively: CRex3-F: 5′-CAGGACTGATGAAAGCAAGC-3′ (SEQ ID NO:6) and Crex3-R: 5′-GCTTATCCATAGCTGTAACAACG-3′ (SEQ ID NO:7); CRex4-F: 5′-GAGACAGTAGGCTGTTCC-3′ (SEQ ID NO:8) and CRex4-R: 5′-CCACAAGCAGTTTCACTACC-3′ (SEQ ID NO:9). PCR fragments were Sanger sequenced using the same forward primers, and sequencing traces were uploaded for analysis with the Synthego ICE analysis web tool to assess editing outcomes.
Stage 45 Xenopus embryos were paralyzed with benzocaine or tricaine and scored with a light stereomicroscope. Cardiac looping was determined by position of the outflow tract; D-loop: rightward, L-loop: leftward; A-loop: midline. Normal intestinal looping was scored as counter-clockwise rotation of the gut, while abnormal intestinal looping was scored as completely inverse gut rotation (clockwise) or complete lack of looping (un-looped). While a completely inverted gut rotation is clearly an abnormality of LR patterning, an unlooped gut is less clear so the present study only considered an unlooped gut as abnormal situs when combined with abnormal placement (left-sided or midline) of the gall bladder. To quantify total abnormal organ situs, each tadpole was counted only once, regardless of whether multiple organs were affected.
Digoxigenin-labeled antisense probes for pitx2 (TNeu083k20), dand5/coco (TEgg007d24), myoD (Tneu017H11), myf5 (TGas127b01), tbxt (TNeu024F07), foxj1 (Tneu058M03), ectodermin (TNeu104j16), foxIla (Tgas002H16), mixer (TGas105b05), vegT (TGas066f22), gsc (TNeu077f20), xnr3 (Tgas011k18), vent2 (BG885317), oct25 (TGas051h05), oct60 (IMAGE: 7526158), oct91 (IMAGE: 7575764), vent1 (BG487195), sox2 (Tgas061h22), cytokeratin (IMAGE:6991625) and sox1713 (BG886038) were in vitro transcribed using T7 High Yield RNA Synthesis Kit (E20405) from New England Biolabs. In order to generate a full-length antisense probe for X. tropicalis kcnh6, kcnh6 cDNA was cloned from stage 45 tadpole whole mRNA using primers xtkcnh6-F: 5′-ATGTTTATCCTAAACTTCGAGGACC-3′ (SEQ ID NO:10) and xtkcnh6-R: 5′-CTAACTTCCTGGAAGACCTGGG-3′ (SEQ ID NO:11) (XM_012952904.1). It was noted that kcnh6 had been misannotated as kcnh2 in the v7.1 model of the X. tropicalis genome (The present study used NCBI Annotation XP_012808358.2 to identify KCNH6). Embryos were collected at the desired stages, fixed in MEMFA for 1-2 h at room temperature (RT) and dehydrated in 100% ethanol. GRPs were dissected post fixation and prior to dehydration to detect dandy. To detect putative gene expression in the prospective endoderm (mixer, vegT, kcnh6) gastrula stage embryos were bisected to facilitate better probe access. Briefly, whole mount in situ hybridization of digoxigenin-labeled antisense probes was performed overnight, the labelled embryos were then washed, incubated with anti-digoxigenin-AP Fab fragments (Roche 11093274910), and signal was detected using BM-purple (Roche 11442074001).
Normal embryonic medium is 1/9× modified Ringer's (MR) containing 11 mM NaCl, 0.2 mM KCl, 0.2 mM CaCl2), 0.1 mM Mg2Cl and 0.55 mM HEPES. To allow Ergtoxin to penetrate the embryos, the present study manually removed the vitelline envelope of stage 8 embryos and incubated embryos in 1/9×MR containing 50 nM Ergtoxin (Alomone STE-450) until stage 12. Embryos were then transferred back into 1/9×MR lacking Ergtoxin to develop until stage 45 in order to score organ situs. Barium chloride was applied into the medium at 20 mM and embryos were thoroughly rinsed in 1/9×MR after each incubation period for further development in Ba2+-free medium. For extracellular K+ manipulations, the KCl concentration in 1/9×MR was modified from 0.2 mM (normal) to 20 mM (high) or 0 mM (low). The ionophore valinomycin (ACROS) and L-type VGCC blocker Nifedipine (ACROS) were diluted in DMSO as stock solutions and applied to embryos in 1/9×MR at 2 nM and 10 μM respectively. Treatments performed during gastrulation were applied from stage 8 through stage 12, and embryos were then rinsed thoroughly and returned into 1/9×MR. For rapamycin, the present study created a standard stock solution of 50 mg/ml in DMSO. The stock solution was diluted 1:2500 in the appropriate embryonic media (final 20 μg/ml). Embryos were treated at stage 7 and then fixed at stage 11 for in situ hybridization.
Embryos were fixed at stage 17 in 4% paraformaldehyde-PBS for 2 h at RT, washed in PBS, and then dissected to obtain GRPs. GRPs were permeabilized for 30 min at RT using 0.1% Triton-PBS (PBST), then blocked in 1% BSA-PBST for 1 h at RT and incubated in primary antibodies diluted in 1% BSA-PBST overnight at 4° C. (anti-myoD LsBio C143580-100 or anti-acetylated tubulin Sigma T-6793). GRPs were then washed in PBST for 30 min and then incubated with secondary antibodies in 1% BSA-PBST for 1h at RT. Phalloidin (1:50; Molecular Probes) and Hoechst 33342 (1:1000; Molecular Probes) were diluted into the secondary antibody solution. Images were acquired using a ZEISS 710 laser scanning confocal microscope.
For recordings, devitellinized, stage 10 kcnh6 or control MO injected embryos were mounted into non-toxic clay with their animal pole exposed and covered with 1/9×MR. To investigate the resting potential, animal pole cells were impaled with a high-impedance (˜70 Me), sharp microelectrode filled with 3 M KCl for intracellular recordings. The recordings were made using an Axon 200B amplifier and digitized using a Digidata 1320 digitizer. Jclamp software for Windows was used in current clamp mode. All electrodes were zeroed just before entry into the cells.
For the series of intracellular recordings in high K+ and choline treated embryos, stage 8-9 embryos were impaled similarly with an electrode of −40 Me. These recordings were made using a HEKA EPC10 amplifier. The present study used HEKA PatchMaster v2x67 software for Windows. All electrodes were zeroed just before entry into the cells.
GCaMP6 and mCherry mRNAs were mixed and injected into embryos at the one-cell stage. Half of these embryos were then injected with kcnh6 MO and the other half with control MO, still at the one-cell stage. Embryos were transferred at stage 10 into the round wells of a press-to-seal silicone isolator (Sigma 53685) mounted between two cover slips in 2% Methylcellulose- 1/9×MR. GcaMP6 and mCherry fluorescence was then captured for 20s (1 frame per second) via time lapse in the whole animal pole of each embryo with a 20× objective of an LSM710 confocal microscope using identical acquisition settings across Control MO and kcnh6 MO embryos. Time lapse recordings were conducted randomly and in an unbiased manner in regard to presence and intensity of calcium transients. However, all embryos did display transient increases in GcaMP6 fluorescence, varying in intensity and spreading to multiple cells. The frames of each recording were sorted to identify the calcium transient peak (in area), and GcAMP6 fluorescence intensity was quantified at peak as a ratio to mCherry in mCherry+ cells. The maximum Ca′ transient area was calculated by demarcating in Fuji the GCaMP6(+) vs GcaMP6(−) area of the animal pole at transient peak. To avoid mosaicism artifacts, only embryos with even, non-mosaic mCherry expression across the entire animal pole were considered. To avoid embryonic stage dependent fluctuations in Ca′ transient size, the present study verified each embryo for stage by progression of blastopore closure and alternated recordings of control and kcnh6 MO embryos. Of note, there were no notable differences in mCherry expression between Control MO and kcnh6 MO embryos.
After manually removing the vitelline envelope of stage 9 or 12 embryos, animal caps were excised and placed on agarose coated dishes in 1/9×MR solution. Caps were then directly placed into agarose coated wells of a 96-well plate in ⅓×MR containing 0.1% BSA and cultured without activin to allow for differentiation into epidermis, with low (20 ng/ml) activin to induce mesoderm, or high activin (200 ng/ml) to induce endoderm. Explants were raised at 25° C. until reaching the equivalent of stage 18 (monitored in whole embryos of the same batch), then fixed in 4% paraformaldehyde, washed in PBS, bleached to eliminate pigmentation (0.5×SSC, 5% formamide, 1.2% H2O2), and then processed by in situ hybridization as described above.
For RNA-Seq, embryos were kept at 25° C. either in 1/9×MR or in 10 mM KCl solution, and 10 embryos were harvested per time point and condition every 30 min starting at stage 8 and concluding at stage 13. Samples were immediately frozen and kept at −80° C. until homogenized in 100 μl Trizol spiked with ERCC RNA Spike-In Mix. 10 μl ERCC RNA Spike-In Mix (Thermo Fisher Scientific) were first diluted into a final volume of 870 μl DEPC water and then further diluted 1:10 into Trizol, which was used to homogenize the samples. Total RNA was purified from the embryo Trizol homogenates according to the manufacturer's recommendations. After isopropanol precipitation, RNAs were resuspended in DEPC water and any contaminating genomic DNA was removed by overnight precipitation in 5M LiCl at 4° C. RNA was subsequently pelleted and washed twice with 70% ethanol. All RNAs were resuspended in DEPC water (2 μl/embryo), and finally, RNA quality was verified by Bioanalyzer. All libraries were sequenced with 100-bp paired-ends on an Illumina NovaSeq6000.
In experiments where embryos were evaluated for phenotypes and scored (gastrulation, left-right patterning, in situ hybridizations) the present study carried out three to five biological replicates and Fisher's exact test to evaluate statistical significance. The animal cap experiment was performed twice with a total score of four to eight animal caps per experiment. For the calcium transient analyses, data was collected from three to five embryos in each experiment in three independent experiments, and statistical analyses on GCaMP/mCherry fluorescence intensity as well as Ca2+ transient area were performed using student's t-test. For whole cell electrophysiological recordings, three to five embryos (two cells each) were examined for their membrane potential and statistical significance was tested by student's t-test. Graphs were designed using GraphPad Prism software. Models were created with BioRender.com.
hESC Culture
hESCs were grown in mTeSR1 (STEMCELL Technologies) in tissue culture dishes coated with Matrigel (Corning; 1:200 in DMEM/F12) and kept at 37° C., 5% CO2. The cell lines used were ESI017 (ESIBIO) and H9. Cells were routinely passaged using dispase (STEMCELL Technologies) and tested for mycoplasma contamination and found negative. For rapamycin experiments, cells were grown in MEF-conditioned HUESM media supplemented with 20 ng/ml bFGF with or without 100 nM rapamycin, which the present study found to increase the survival of rapamycin treated cells compared to cells grown in mTeSR1.
hESC Treatments and Differentiation
Cells were dissociated with accutase and seeded onto 8 well imaging slides (ibidi 80826) at a density of 4-6×104/cm2. Cells were seeded and maintained in Rock-inhibitor Y27672 (MCE; 10 μM) to increase survival and the uniformity of response. Treatments with 1 mM BaCl2 or 10 or 25 nM Ergtoxin or 100 nM rapamycin were initiated 4 hours after seeding. Differentiation was initiated 24 hours after seeding where indicated. To initiate differentiation, the media was replenished with/without BaCl2 or Ergtoxin and treated with the indicated growth factors or small molecules. Cells were incubated for the indicated times without media change before fixation.
Immunofluorescence of hESCs
Cells were fixed for 30 min in 4% paraformaldehyde, rinsed twice with DPBS (without Ca2+ and Mg2+, denoted DPBS−/−), and blocked for 30 min at room temperature. The blocking solution contained 3% donkey serum and 0.1% Triton X-100 in 1×DPBS−/−. After blocking, the cells were incubated with primary antibodies at room temperature for 2 hours. Antibodies and concentrations are listed below. Cells were washed three times with DPBST (1×DPBS−/− with Tween 20) and incubated with secondary antibodies (AlexaFluor 488 A21206, AlexaFluor 555 A31570 and A21432, and AlexaFluor 647 A31571, Thermo Fisher; 1:500) and DAPI for 30 min at room temperature. After secondary antibody incubation, samples were washed in DPBST and then DPBS at room temperature. The antibodies used for the experiments are also listed in
hESC Imaging and Analysis
Images were acquired using a 20×, NA 0.75 objective on an Olympus IX83 inverted epifluorescence microscope or an Olympus/Andor spinning disk confocal microscope. Cell segmentation was performed using ilastik software. This segmentation was cleaned (to remove debris and to separate merged cells) and mean nuclear protein intensities as well as standard errors were quantified using a custom MATLAB code. Nuclear intensities were normalized by DAPI to correct for intensity variation due to optics. Code is available at https://github.com/warmflasha/celltracker.
qPCR
For qPCR, hESCs were grown with or without ErgToxin (25 nM) for the indicated times. RNA collection and DNase treatment were performed using the RNAqueous®-Micro Total RNA Isolation Kit (AM1931) and cDNA was synthesized with the SuperScript Vilo cDNA Synthesis Kit (Fisher Scientific 11754-050). qPCR measurements were collected using SYBR Green reagent (LifeTech-4367659) on a Step OnePlus instrument (Applied Biosciences). Data were normalized using the housekeeping gene GAPDH. Primers for qPCR were: OCT4: 5′-caagctcctgaagcagaagag-3′ (SEQ ID NO:12), 5′-ccaaacgaccatctgccgcttt-3′ (SEQ ID NO:13), SOX2: 5′-ccatgcaggttgacaccgttg-3′ (SEQ ID NO:14), 5′-tcggcagactgattcaaataata-3′ (SEQ ID NO:15), NANOG: 5′-tgggatttacaggcctgagcca-3′ (SEQ ID NO:16), 5′-aagcaaagcctcccaatcccaaa-3′ (SEQ ID NO:17), GAPDH: 5′-caccgtcaaggctgagaacg-3′ (SEQ ID NO:18), 5′-gccccacttgattttggagg-3′ (SEQ ID NO:19).
Stranded paired end 100 bp RNA-seq reads were aligned to the Xt9.1 genome combined with ERCC spikes using STAR1 and quantified as transcripts per million (TPM) for each isoform with RSEM2 using the RSEM-STAR pipeline, with additional options “—seed 1618 —calc-pme —calc-ci —estimate-rspd —paired-end”. Using the ERCC spikes the present study identified a batch-dependent GC bias where AT-rich transcripts were preferentially lost as compared to GC-rich transcripts (
s to its known concentration ci:
The present study used the GLM.jl (https://github.com/JuliaStats/GLM.jl) in the Julia language to apply this model and add a pseudocount of 2 to all dinucleotide frequencies. As the GC effect varies between UIC and high K+ samples (Ext Data
The present study first filtered 34,192 quantified genes to find those with sufficient temporal expression for further analysis, the present study selected genes that had runs of 6 consecutive samples with uncorrected TPM>0.4. This resulted 13,310 from which the present study excluded a further 162 genes which where excessively altered by the above-described correction procedure, these had log 2 fold changes between corrected or uncorrected quantifications outside of the interval (−2.5, 4.5). After dinucleotide correction and filtering the present study found excellent concordance between samples, with minimal evidence of outlying samples, by Spearman Correlation comparisons and principal components analysis (PCA) (
To determine genes temporally differentially expressed the present study used Gaussian process (GP) regression. All GP regression was performed with GaussianProcesses.j1 (https://github.com/STOR-i/GaussianProcesses.j1; https://arxiv.org/abs/1812.09064). Due to the overdispersed nature of RNA-seq count data, the present study applied a variance stabilising transform that puts all genes on the same scale: ysi=√{square root over (α+βxsi/mi)}, with xsi the dinucleotide corrected abundance of gene i in sample s, mi the maximum xsi over all samples, and α=1, β=1000. The present study then performed exact GP regression (GP prior and a Gaussian likelihood) with Matern52 kernel, the present study optimized the three associated hyperparameters: σf2 or the signal variance, τ the timescale (this parameter is commonly referred to as the lengthscale ), and σf2 the sample noise variance. Parameters are selected by optimising marginal log-likelihood with parameters in log space: log σf, log τ, log σn, and to ensure physiologically reasonable values for each the present study placed Gaussian priors,
(μ, σ) over each of these variables respectively
(1.4, 4.0),
(1.2, 1.0),
(1.0, 0.75). Finally, the present study reported GP median and 95% confidence intervals through the inverted data transformation {circumflex over (x)}si=mi(ŷsi1−α)/β and set {circumflex over (x)}si=0 for ŷsi<√{square root over (α)}.
To determine temporal differential expression, the present study calculated a marginal likelihood ratio for whether separate GP models for UIC and high K+ or a single GP model for all data combined is preferred. If Lu and Lk are the marginal log-likelihoods for UIC and high K+ respectively, and Luk is the marginal log-likelihood for a single regression through UIC and high K+ together. Then the present study calculate log-likelihood ratio LR=Lu+Lk−Luk of evidence in favour of two models (essentially that the UIC and High K+ have different expression trajectories for a given gene) and determine genes with LR>0 as temporally differentially expressed. This resulted in 5144 differentially expressed genes, with 4043 activated and 1101 repressed (
To determine sets of differentially expressed genes with similar trajectories, the present study applied K-means clustering to activated and repressed genes independently. The present study define a gene as activated if Gaussian process median for High K+ exceeds UIC on average, and repressed if it does not, the present study found no genes for which the mean of High+ and UIC differences was zero. The present study cluster UIC and High K+ genes by taking Gaussian process medians and normalising by the maximum value experience by UIC or High K+. The present study then clustered both trajectories together employing the kmeans function offered by Clustering.jl (https://github.com/JuliaStats/Clustering.jl) with default settings and random seed 16. To select the cluster number, the present study calculated the silhouette score for activated and repressed clusters for k=2-10. The present study found that the maximal mean silhouette score activated genes was k=3 and for repressed genes was k=2, but that scores were broadly similar for k=2-4 and decreased significantly for k>4, suggesting that k=4 provides a reasonable partition of the data. In line with this the present study explored the clusters from k=2-10, and found that key clusters were not well-resolved for k<4 and that k>4 clusters refined k=4 behaviours. As k>4 did not reveal new behaviours and did not improve gene set enrichments, the present study selected k=4 to cluster activated and repressed genes.
To assess the composition of each cluster the present study performed gene set enrichments using Enrichr. The present study took genes from each cluster with a known Xenopus gene symbol and converted these to human symbols, by removing any “.N” suffix for an integer N (for example, ventx1.1 becomes ventx1) and converting to uppercase. The present study then made the following substitutions to convert certain known Xenopus gene symbols to human where the name of the ortholog has diverged or only a paralog exists: pou5f3→POU5F1, mix1→mixl1, dppa2→DPPA4, lefty→lefty2, ventx1-3→NANOG, mespb→MESP1, sox17a/b→SOX17. The present study removed any duplicate names that arose in this process. The present study calculated enrichments for the following gene sets: KEGG_2019 Human, BioPlanet_2019, WikiPathways_2019 Human, GO_Biological_Process_2018, GO_Molecular_Function_2018, GO_Cellular_Component_2018, ChEA_2016. The present study calculated enrichments for each cluster individually and consecutive combinations of the 4 clusters: 1, 2, 3, 4, 12, 23, 34, 123, 234, 1234. Selected enrichments are given in
To find motifs enriched in the promoters, the present study took the 500 bp upstream of the promoter of the maximally expressed isoform for each gene in the four activated and four repressed clusters, along with a background of the 500 bp upstream of all annotated TSS in the Xt9.1 genome. The present study extracted fasta files for each of these sets of regions, and then used findMotifs.pl from Homer5 to search for known motifs with options: “findMotifs.pl clusterAB.fa fasta outAB -fasta background.fa -nomotif” where A∈{activated, repressed} and B∈{1, 2, 3, 4}. The present study filtered results to select best matching motifs from related families, namely the present study collapsed all ETS motifs to the canonical Homer ETS promoter motif; all SP and KLF motifs to SP1; SOX motifs to SOX2; all HOX motifs to HOXD13 (the highest scoring HOX); the present study represented all GFY and Ronin matches as ZNF143 (for which the motifs overlap); and the present study excluded motif annotated as PRDM10, due to low confidence in the motif. The motif annotated as ATF1 is an example of the cAMP response element (CRE) bound by CREB factors including ATF1, the present study labels this as CRE/ATF1. The top 16 motif enrichments are given in
To calculate CRE and ETS motif enrichment for mTOR and pluripotency genes, the present study took genes annotated with the terms mTOR signaling pathway and Signaling pathways regulating pluripotency of stem cells from KEGG_2019 Human as provided by Enrichr4 that are activated in high K+ (LR>0) and are present in clusters A1 and A2. The resulting genes were subjected to the same promoter analysis, using Homer to calculate the occurrence of the maximal ATF/CRE family motif and the ETS motif in these promoters and the background set to report Fisher Exact test p-values and Odds Ratios.
In some aspects, the present invention is directed to the following non-limiting embodiments:
Embodiment 1: A method of maintaining pluripotency in a cell, comprising at least one of the following: reducing the membrane potential of the cell; activating a voltage gated calcium channel on the plasma membrane of the cell; or increasing the calcium ion concentration in the cell.
Embodiment 2: The method of Embodiment 1, wherein reducing the membrane potential of the cell comprises at least one of the following: subjecting the cell to an extracellular environment having a high concentration of potassium ions; inhibiting a potassium channel on the plasma membrane of the cell; or contacting the cell with a potassium selective ionophore.
Embodiment 3: The method of any one of Embodiments 1-2, wherein reducing the membrane potential of the cell comprises subjecting the cell to an extracellular environment having a concentration of potassium ions of about 0.5 mM or higher, such as about 0.75 mM or higher, about 1 mM or higher, about 2 mM or higher, about 3 mM or higher, about 4 mM or higher, about 5 mM or higher, about 7.5 mM or higher, about 10 mM or higher, about 12.5 mM or higher, about 15 mM or higher, about 20 mM or higher, about 25 mM or higher, about 30 mM or higher, about 40 mM or higher, or about 50 mM or higher.
Embodiment 4: The method of any one of Embodiments 1-2, wherein reducing the membrane potential of the cell comprises inhibiting a potassium channel on the plasma membrane of the cell, and wherein the potassium channel comprises an inwardly-rectifying voltage gated potassium channel.
Embodiment 5: The method of Embodiment 4, wherein the inwardly-rectifying voltage gated potassium channel comprises potassium voltage-gated channel subfamily H member 6 (KCNH6).
Embodiment 6: The method of any one of Embodiments 2 and 4-5, wherein inhibiting the potassium channel on the plasma membrane of the cell comprises contacting the cell with a potassium channel inhibitor.
Embodiment 7: The method of Embodiment 6, wherein the inhibitor of the potassium channel comprises barium ions or an Ergtoxin.
Embodiment 8: The method of any one of Embodiment 1-2, wherein reducing the membrane potential of the cell comprises contacting the cell with a potassium selective ionophore comprising valinomycin, BME 44 (2-Dodecyl-2-methyl-1,3-propanediyl bis[N-[5′-nitro(benzo-15-crown-5)-4′-yl]carbamate]), or BB15C5 (Bis[(benzo-15-crown-5)-4′-ylmethyl] pimelate).
Embodiment 9: The method of Embodiment 1, wherein the voltage gated calcium channel is an L-type calcium channel or a T-type calcium channel.
Embodiment 10: The method of any one of Embodiments 1-9, wherein the cell is a stem cell.
Embodiment 11: The method of any one of Embodiments 1-10, wherein the cell is an embryonic stem cell.
Embodiment 12: The method of any one of Embodiments 1-11, wherein the cell is in an organism, a cultured primary cell, or a cultured cell line.
Embodiment 13: The method of any one of Embodiments 1-12, wherein the cell is from a vertebrate origin, optionally from a mammalian origin and/or a human origin.
Embodiment 14: A composition, comprising: a culture medium; and a pluripotent cell, wherein potassium ions in the culture medium are present in a concentration of about 0.5 mM or higher, and wherein a pluripotency of the pluripotent cell is maintained by the concentration of potassium ions in the culture medium.
Embodiment 15: The composition of Embodiment 14, wherein the concentration of the potassium ions in the culture medium reduces the membrane potential of the cell, thereby maintaining the pluripotency of the pluripotent cell.
Embodiment 16: The composition of any one of Embodiments 14-15, wherein the culture medium comprises: at least one inorganic ion selected from a sodium ion, a potassium ion, a calcium ion, a magnesium ion, a chloride ion, a sulfate ion, a carbonate ion, a bicarbonate ion, a phosphate ion, a phosphate monobasic ion, a phosphate dibasic ion; an amino acid; and a vitamin.
Embodiment 17: The composition of claim 16, wherein the amino acid comprises at least one selected form the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; the vitamin comprises at least one selected from the group consisting of pantothenate, choline, folic acid, inositol, nicotinamide, pyridoxine, riboflavin, and thiamine; the culture medium further comprises a carbohydrate; or the culture medium further comprises one or more selected form the group consisting of pyruvate, lipoic acid, biotin, a buffering agent, and a pH indicator.
Embodiment 18: The method of any one of Embodiments 14-17, wherein the pluripotent cell is a stem cell or an embryonic stem cell.
Embodiment 19: The method of any one of Embodiments 14-18, wherein the pluripotent cell is from a vertebrate origin, a mammalian origin, or a human origin.
Embodiment 20: The method of any one of Embodiments 14-19, wherein the pluripotent cell does not have the cell potency to develop in to a human.
The foregoing outlines features of several embodiments so that those skilled in the art may better understand the aspects of the present disclosure. Those skilled in the art should appreciate that they may readily use the present disclosure as a basis for designing or modifying other processes and structures for carrying out the same purposes and/or achieving the same advantages of the embodiments introduced herein. Those skilled in the art should also realize that such equivalent constructions do not depart from the spirit and scope of the present disclosure, and that they may make various changes, substitutions, and alterations herein without departing from the spirit and scope of the present disclosure.
The present application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63/388,380, filed Jul. 12, 2022, which is incorporated herein by reference in its entirety.
This invention was made with government support under 1HL149746-01A1 awarded by National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63388380 | Jul 2022 | US |
