Patent Application
20180044637
Stem cells have numerous uses, such as for research and for treatment of a variety of diseases. However, numerous factors (e.g., growth conditions, oxygen levels, cell age etc.) can contribute to the effectiveness of stems cells and alter their propensity for differentiation. Additionally, the properties of the stem cell can have an impact on the ability for their use in a variety of manner, such as, for example, transplantation. Accordingly, some embodiments of the present invention include methods for growing stem cells in cell medium that address one or more of the issues. Additional embodiments of the invention are also discussed herein.
Some embodiments of the present invention include methods for growing first stem cells comprising growing the first stem cells in a cell medium having a pH of from about 6.6 to about 7.2, and recovering second stem cells. In certain embodiments, the pH of the cell medium is from about 6.7 to about 7.1, or is from about 6.8 to about 7.0, or is about 6.9. In still other embodiments, the first stem cells are grown ex vivo. In yet other embodiments, the first stem cells are human stem cells, mouse stem cells, rat stem cells, primate stem cells, or mammalian stem cells. And in yet other embodiments, the first stem cells are somatic stem cells, tissue-specific stem cells, hematopoietic stem cells (HSC), stem cells that express CD34, mammary stem cells, intestinal stem cells, mesenchymal stem cells, endothelial stem cells, neural stem cells, olfactory adult stem cells, neural crest stem cells, or testicular stem cells. In some embodiments, there is an increase in an amount of chimera cells in peripheral blood, when the second stem cells are competitively transplanted into a recipient, when compared to transplanted stem cells that were grown in cell medium of pH about 7.4 (e.g., an increase of at least about 20%). In other embodiments, the second stem cells have a decreased percentage of cells that are in the S phase when compared to stem cells grown in cell medium of pH about 7.4 (e.g., a decrease of at least about 20%). In still other embodiments, second stem cells have a decreased amount of reactive oxygen species compared to stem cells grown in cell medium of pH about 7.4 (e.g., a decrease of at least about 20%). In yet other embodiments, the second stem cells have a decreased level of CD34 expression when compared to stem cells grown in cell medium of pH about 7.4 (e.g., a decrease of at least about 20%). In some embodiments. the second stem cells have a decreased volume of the stem cell when compared to stem cells grown in cell medium of pH about 7.4 (e.g., a decrease of at least about 20%). In certain embodiments, the second stem cells have a decreased amount of H4K16ac in the stem cell when compared to stem cells grown in cell medium of pH about 7.4 (e.g., a decrease of at least about 20%). In other embodiments, the second stem cells have an increased glutathione expression in the stem cell when compared to stem cells grown in cell medium of pH about 7.4 (e.g., an increase of at least about 20%). In some embodiments, the second stem cells have a decreased expression of one or more proteases (e.g., the one or more proteases are cathepsin G, granzyme B, or both) in the stem cell when compared to stem cells grown in cell medium of pH about 7.4 (e.g., a decrease of at least about 20%). In still other embodiments, the first stem cells are taken from a mouse with an age of at least about 2 months, at least about 10 months, or at least about 18 months. In other embodiments, the first stem cells are taken from a human with an age of at least about 10 years, at least about 50 years, or at least about 65 years. In yet other embodiments, recovering comprises one or more of centrifuging, filtering, or washing.
Some embodiments of the invention include methods for transplanting comprising (a) growing first stem cells in a cell medium having a pH of from about 6.6 to about 7.2, to provide second stem cells and (b) transplanting the second stem cells into a recipient. In certain embodiments, the recipient is a mammal, rodent, primate, monkey, human, canine, feline, porcine, mouse, rabbit, or rat. In other embodiments, the transplanting of the second stem cells in the recipient occurs in the bone, bone marrow, blood, skin, brain, or mammary. In yet other embodiments, the pH of the cell medium is from about 6.8 to about 7.0 or is about 6.9. In some embodiments, the first stem cells are grown ex vivo. In certain embodiments, the first stem cells are human stem cells, mouse stem cells, rat stem cells, primate stem cells, or mammalian stem cells, or are somatic stem cells, tissue-specific stem cells, hematopoietic stem cells (HSC), stem cells that express CD34, mammary stem cells, intestinal stem cells, mesenchymal stem cells, endothelial stem cells, neural stem cells, olfactory adult stem cells, neural crest stem cells, or testicular stem cells. In some embodiments, the first stem cells are hematopoietic stem cells (HSC) or stem cells that express CD34. In other embodiments, the method further comprises recovering the second stem cells before step (b).
Other embodiments of the invention are also discussed herein.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the description of specific embodiments presented herein.
Some embodiments of the invention include methods comprising growing first stem cells in a cell medium having a pH of from about 6.6 to about 7.2, to provide second stem cells. Some embodiments of the invention include methods for transplanting comprising (a) growing first stem cells in a cell medium having a pH of from about 6.6 to about 7.2, to provide second stem cells and (b) transplanting the second stem cells into a recipient. In certain embodiments, the pH of the cell medium can be about 6.6, about 6.65, about 6.7, about 6.75, about 6.8, about 6.85, about 6.9, about 6.95, about 7.0, about 7.05, about 7.1, about 7.15, about 7.2, from about 6.6 to about 7.2, from about 6.7 to about 7.2, from about 6.7 to about 7.1, from about 6.7 to about 7.0, from about 6.8 to about 7.1, or from about 6.8 to about 7.0. The pH of the cell medium can be measured using any suitable method including but not limited to using a 0.6 mm combination pH probe (Specialty Sensor LLC USA) or using a pre-calibrated standard pH Meter (Toledo, USA). The pH of the cell medium can be adjusted using any suitable technique or means including but not limited to one or more of addition of a strong acid (e.g., HCl), addition of a weak acid (e.g., citric acid), additional of a strong base (e.g., NaOH), additional of a weak base (e.g., alanine), additional of one or more buffers (HEPES), additional of macromolecules that increase the pH, or addition of macromolecules that decrease the pH.
In other embodiments, the method further comprises recovering the second stem cells. Recovering can include any suitable method or means to recover the second stem cells, such as but not limited to one or more of purifying, isolating, centrifuging, filtering, or washing (e.g., 1, 2, 3, 4, 5, 6, or 7 times, with the same or different washing solutions) the second stem cells.
In some embodiments, the recipient can be an animal that is the same or different as the origin of first stem cells. In other embodiments, the recipient can be any suitable animal, such as but not limited to mammals, rodents, primates, monkeys, humans, canine, feline, porcine, mice, rabbits, or rats. In yet other embodiments, the recipient can have the second stem cells transplanted in any suitable place in the recipient's body, such as but limited to an organ, bone, bone marrow, blood, lymph node, brain, liver, kidney, testicle, skin, intestine, stomach, or mammary. In certain embodiments, the transplant can be an autologous transplant, an allogeneic transplant, an umbilical cord transplant (e.g., blood), a parent-child transplant, a haplotype mismatched transplant, or a xenotransplant.
In some embodiments, the first stem cells are grown ex vivo (e.g., living cells or tissues taken from an organism and cultured in one or more laboratory devices (e.g., incubators, flasks, Petri dishes, roller bottles, or multi-well plates)). In certain embodiments, the first stem cells are grown under any suitable technique or condition such as but not limited to one or more of using sterile conditions, using controlled temperature in the cell environment (e.g. from about 36° C. to about 37° C.), using controlled carbon dioxide (e.g., from about 5 to about 7% CO2) in the cell environment (e.g., in the hood or incubator), using controlled humidity in the cell environment (e.g., in the hood or incubator), using a cell culture hood (e.g., class I, class II or class III), using aseptic technique, using adherent culture techniques, using suspension culture techniques, using tissue culture techniques, or any other suitable technique or condition.
The cell medium can be any suitable cell medium, including but not limited to Gibco StemPro-34 SFM (from Thermo Fischer), PromoCell Hematopoietic Progenitor Cell (HPC) Expansion Medium DXF (from PromoCell), Iscove modified Dulbecco medium (IMDM; Cellgro, Catalog #21-020-CV), Stem-Span medium from stem cell technologies, and media from Miltenyi biotec (e.g., a StemMACS media or a Cytomix media). In some embodiments, the cell media is serum-free or the cell media comprises serum. In some embodiments, the growth conditions (e.g., the cell medium) are not identical to (e.g., are not substantially similar to) growth conditions (e.g., the composition of cell nutrient environment) found in vivo or found in the environment from where the first stem cells came or originated. In other embodiments, the cell media is stabilized (e.g., stabilizing the pH) in an incubator without cells; the amount of time for stabilization can be any suitable time including but not limited to for about 1 hr (hour), about 2 hr, about 3 hr, about 4 hr, about 8 hr, about 12 hr, about 16 hr, about 24 hr, about 28 hr, about 32 hr, about 36 hr, about 40 hr, about 44 hr, about 48 hr, about 52 hr, about 56 hr, about 60 hr, about 64 hr, about 68 hr, about 72 hr, about 76 hr, about 80 hr, about 84 hr, about 88 hr, about 92 hr, about 96 hr, about 3.5 days, about 4 days, about 4.5 days, about 5 days, about 5.5 days, about 6 days, from about 1 hr to about 6 days, no more than about 6 days, at least about 1 hr, from about 12 hr to about 4 days, or from about 24 hr to about 3 days.
The first stem cells that can used in the methods described herein (e.g., growing in a cell medium having a pH of about 6.6 to about 7.2) can be any suitable stem cells, such as but not limited to somatic stem cells, tissue-specific stem cells, hematopoietic stem cells (HSC, also referred to as hemocytoblasts), stem cells that express CD34, mammary stem cells, intestinal stem cells, mesenchymal stem cells, endothelial stem cells, neural stem cells, olfactory adult stem cells, neural crest stem cells, or testicular stem cells. The first stem cells (e.g., HSCs) can come from any suitable animal, such as but not limited to mammals, rodents, primates, monkeys, humans, canine, feline, porcine, mice, rabbits, or rats. In some instances, the first stem cells (e.g., HSCs) can be any suitable stem cells, such as but not limited to primary stem cells, secondary stem cells, cells from an animal (e.g., within any suitable time such as but not limited to within about 1 hour, within about 2 hours, within about 4 hours, within about 8 hours, within about 12 hours, within about 24 hours, or within about 48 hours), or cells thawed from a freezer (e.g., where the thawing took place within any suitable time such as but not limited to within about 1 hour, within about 2 hours, within about 4 hours, within about 8 hours, within about 12 hours, within about 24 hours, or within about 48 hours).
In some embodiments, the first stem cells (e.g., HSCs) are taken (e.g., harvested) from an animal (e.g., a human or a mouse) with an age of about 0.1 mo. (month), about 1 mo., about 2 mo., about 3 mo., about 4 mo., about 5 mo., about 6 mo., about 7 mo., about 8 mo., about 9 mo., about 10 mo., about 11 mo., about 12 mo., about 13 mo., about 14 mo., about 15 mo., about 16 mo., about 17 mo., about 18 mo., about 19 mo., about 20 mo., about 21 mo., about 22 mo., about 23 mo., about 24 mo., about 25 mo., about 26 mo., about 27 mo., about 28 mo., about 29 mo., about 30 mo., about 31 mo., about 32 mo., about 33 mo., about 34 mo., about 35 mo., about 36 mo., about 1 yr., about 1.5 yr., about 2 yr., about 2.5 yr, about 3 yr., about 3.5 year, about 4 yr., about 4.5 yr., about 5 yr., about 7.5 yr., about 10 yr., about 15 yr., about 20 yr., about 25 yr., about 30 yr., about 35 yr., about 40 yr., about 45 yr., about 50 yr., about 55 yr., about 60 yr., about 65 yr., about 70 yr., about 75 yr., about 80 yr., about 85 yr., about 90 yr., about 95 yr., about 100 yr., about 150 yr., no more than about 150 yr., no more than about 100 yr., no more than about 50 yr., no more than about 20 yr., no more than about 5 yr., no more than about 20 mo., no more than about 18 mo., no more than about 5 mo., no more than about 4 mo., no more than about 1 mo., at least about 0.1 mo., at least about 1 mo., at least about 2 mo., at least about 4 mo., at least about 5 mo., at least about 10 mo., at least about 15 mo., at least about 18 mo., at least about 2 yr., at least about 10 yr., at least about 20 yr., at least about 35 yr., at least about 50 yr., at least about 65 yrs., at least about 75 yr., at least about 90 yr., from about 0.1 mo. to about 150 yr., from about 1 mo. about 150 yr., from about 5 mo. to about 100 yr., from about 5 mo. to about 50 yr., from about 5 mo. to about 10 yr., from about 5 mo. to about 24 mo., from about 10 mo. to about 100 yr., from about 10 mo. to about 50 yr., from about 10 mo. to about 10 yr., from about 10 mo. to about 24 mo., from about 15 mo. to about 100 yr., from about 15 mo. to about 50 yr., from about 15 mo. to about 10 yr., from about 15 mo. to about 24 mo., from about 10 yr. to about 100 yr., from about 10 yr. to about 50 yr., from about 20 yr. to about 100 yr., from about 20 yr. to about 50 yr., from about 30 yr. to about 100 yr., from about 30 yr. to about 50 yr., from about 50 yr. to about 100 yr., or from about 75 yr. to about 100 yr.
The first stem cells (e.g., HSCs) can be grown in the cell medium for any suitable amount of time including but not limited to for about 1 hr (hour), about 2 hr, about 3 hr, about 4 hr, about 8 hr, about 12 hr, about 16 hr, about 24 hr, about 28 hr, about 32 hr, about 36 hr, about 40 hr, about 44 hr, about 48 hr, about 52 hr, about 56 hr, about 60 hr, about 64 hr, about 68 hr, about 72 hr, about 76 hr, about 80 hr, about 84 hr, about 88 hr, about 92 hr, about 96 hr, about 3.5 days, about 4 days, about 4.5 days, about 5 days, about 5.5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 2.5 weeks, about 3 weeks, about 3.5 weeks, about 4 weeks, about 1.5 months, about 2 months, about 2.5 months, about 3 months, about 4 months, about 5 months, about 6 months, from about 1 hr to about 6 months, no more than about 6 months, at least about 1 hr, from about 12 hr to about 4 months, or from about 24 hr to about 3 months. In some embodiments, the cell medium is refreshed (e.g., replaced with fresh cell medium of the same type) one or more times during the method (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 10, 15, 20, or 25 times); the cell medium can be refreshed at any suitable time from the start of the method or from the previous cell medium refreshing, and the time between any cell medium refreshing can be the same or different as the time between other cell medium refreshings. The time between cell medium refreshing can be any suitable time including but not limited to a one or a combination of about 1 hr (hour), about 2 hr, about 3 hr, about 4 hr, about 8 hr, about 12 hr, about 16 hr, about 24 hr, about 28 hr, about 32 hr, about 36 hr, about 40 hr, about 44 hr, about 48 hr, about 52 hr, about 56 hr, about 60 hr, about 64 hr, about 68 hr, about 72 hr, about 76 hr, about 80 hr, about 84 hr, about 88 hr, about 92 hr, about 96 hr, about 3.5 days, about 4 days, about 4.5 days, about 5 days, about 5.5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, or about 14 days.
In some embodiments, growing first stem cells in a cell medium having a pH of from about 6.6 to about 7.2 (e.g., from about 6.8 to about 7.0 or about 6.9) can result in modulation of one or more cell properties of second stem cells when that property is compared to that of stems cells grown at a pH of about 7.4 (e.g., in the same or similar cell medium where the only difference or only substantive difference (a substantive difference is one where the given property is changed by no more than about 1%, no more than about 5%, no more than about 10%, or no more than about 15%) in the cell medium is that it has a pH of about 7.4). Modulation can be an increase or a decrease. In some embodiments, one or more of the following cell properties of the second stem cell can, in some instances, be modulated (e.g., as compared to cells grown at a pH about 7.4), but cell properties that can be modulated are not limited to those listed here: (a) an increase in the amount of chimera cells in peripheral blood of a recipient, after second stem cells were competitively transplanted (e.g., a bone marrow transplant, a primary transplant, or a secondary transplant) into the recipient (e.g., a lethally irradiated recipient), where the percentage increase in the amount of chimera cells in peripheral blood can be, for example, about 20%, about 30%, about 40%, about 50%, about 100%, about 120%, about 150%, about 200%, about 250%, about 300%, about 400%, about 500%, about 600%, about 700%, at least about 20%, at least about 50%, or at least about 100%; (b) a decrease in the percentage of second stem cells that are in the S phase of the cell division cycle (i.e., compared to stem cells grown at pH about 7.4), where the decrease can be, for example, about 20%, about 30%, about 40%, about 50%, about 100%, about 120%, about 150%, about 200%, about 250%, about 300%, about 400%, about 500%, about 600%, about 700%, at least about 20%, at least about 50%, or at least about 100%; (c) a decrease in the amount of reactive oxygen species (ROS) (e.g., measured using DCF fluorescence) in the second stem cells (i.e., compared to stem cells grown at pH about 7.4), where the percentage decrease in the amount of ROS can be, for example, about 20%, about 30%, about 40%, about 50%, about 100%, about 120%, about 150%, about 200%, about 250%, about 300%, about 400%, about 500%, about 600%, about 700%, at least about 20%, at least about 50%, or at least about 100%; (d) a decrease in the level of CD34 expression (e.g., in mice) in the second stem cells (i.e., compared to stem cells grown at pH about 7.4), where the percentage decrease in level of CD34 expression can be, for example, about 20%, about 30%, about 40%, about 50%, about 100%, about 120%, about 150%, about 200%, about 250%, about 300%, about 400%, about 500%, about 600%, about 700%, at least about 20%, at least about 50%, or at least about 100%; (e) an increase in stabilization of post-translational Hif1a (hypoxia-inducible factor 1-alpha) in the second stem cells (i.e., compared to stem cells grown at pH about 7.4), such as for example, a lack of change in Hif1a expression (e.g., less than about 70% change, less than about 50% change, less than about 20% change, less than about 10% change, less than about 5% change, less than about 2% change, less than about 1% change, or less than about 0.1% change); (f) a decrease in mitochondrial stress of the second stem cells (i.e., compared to stem cells grown at pH about 7.4), as for example indicated by less elongated mitochondria (i.e., compared to stem cells grown at pH about 7.4) or by a decrease vacuolization (i.e., compared to stem cells grown at pH about 7.4), where the percentage decrease in vacuolization (either in number of vacuoles or in volume occupied by vacuoles) can be, for example, about 20%, about 30%, about 40%, about 50%, about 100%, about 120%, about 150%, about 200%, about 250%, about 300%, about 400%, about 500%, about 600%, about 700%, at least about 20%, at least about 50%, or at least about 100%; (g) a decrease in the intracellular pH (pHi) (e.g., measured via Snarf-1) of the second stem cells (i.e., compared to stem cells grown at pH about 7.4), where the percentage decrease can be, for example, about 20%, about 30%, about 40%, about 50%, about 100%, about 120%, about 150%, about 200%, about 250%, about 300%, about 400%, about 500%, about 600%, about 700%, at least about 20%, at least about 50%, or at least about 100%; (h) a decrease in the volume of the second stem cells (e.g., the mean or the median as, for example, measured by flow cytometry and/or confocal microscopy) (i.e., compared to stem cells grown at pH about 7.4), where the percentage decrease can be, for example, about 20%, about 30%, about 40%, about 50%, about 100%, about 120%, about 150%, about 200%, about 250%, about 300%, about 400%, about 500%, about 600%, about 700%, at least about 20%, at least about 50%, or at least about 100%; (i) a decrease in the amount of H4K16ac (acetylation at histone H4 on lysine 12) in the second stem cells (e.g., using FACS analysis) (i.e., compared to stem cells grown at pH about 7.4), where the percentage decrease can be, for example, about 20%, about 30%, about 40%, about 50%, about 100%, about 120%, about 150%, about 200%, about 250%, about 300%, about 400%, about 500%, about 600%, about 700%, at least about 20%, at least about 50%, or at least about 100%; (j) an increase in the glutathione expression in the second stem cells (i.e., compared to stem cells grown at pH about 7.4), where the percentage increase can be, for example, about 20%, about 30%, about 40%, about 50%, about 100%, about 120%, about 150%, about 200%, about 250%, about 300%, about 400%, about 500%, about 600%, about 700%, at least about 20%, at least about 50%, or at least about 100%; (k) an increase in quiescence and/or in self-renewal (e.g., via a CBFA2T3-related mechanism) in the second stem cells (i.e., compared to stem cells grown at pH about 7.4); or (l) a decrease in expression of one or more proteases (e.g., one or more of cathepsin G or granzyme B) in the second stem cells (i.e., compared to stem cells grown at pH about 7.4), where the where the percentage decrease for one or more proteases can be the same or different and can be, for example, about 20%, about 30%, about 40%, about 50%, about 100%, about 120%, about 150%, about 200%, about 250%, about 300%, about 400%, about 500%, about 600%, about 700%, at least about 20%, at least about 50%, or at least about 100%.
In certain embodiments, the methods disclosed herein can result in second stem cells that have elevated contributions to the hematopoietic system upon transplantation into recipients. In yet other embodiments, the methods disclosed herein can result in second stem cells that have enhanced long-term contributions to the hematopoietic system upon transplantation (e.g., via transplantation into secondary and tertiary recipients). In still other embodiments, the methods disclosed herein can result in a more healthy differentiation pattern upon transplantation of second stem cells (e.g., when aged HSCs are transplanted; aged can be, for example, about 18 months or older for mice, or about 60 years old or older for humans); “more healthy” can include but is not limited to less monocytes/macrophages or more lymphoid cells. In additional embodiments, the methods disclosed herein can result in an enhanced time to neutrophil recovery after transplantation of second stem cells.
In some embodiments, the methods described herein can be part of any suitable method or procedure, such as but not limited to ex vivo manipulation of stem cells (e.g., HSCs), collecting stem cells (e.g., HSCs), collecting and freezing stem cells (e.g., HSCs), thawing stem cells (e.g., HSCs), maintaining ex vivo stems cells (e.g., HSCs) as part of genetically modifying stem cells (e.g., HSCs), transplanting stem cells (e.g., HSCs), transplanting stem cells (e.g., HSCs) where the stem cells (e.g., HSCs) were previously frozen, transplanting stem cells (e.g., HSCs) where the stem cells (e.g., HSCs) were not previously frozen, and research methods for studying stem cells (e.g., HSCs).
The presently-disclosed subject matter is further illustrated by the following specific but non-limiting examples. The following examples may include compilations of data that are representative of data gathered at various times during the course of development and experimentation related to the present invention.
We first set out to determine pH levels within the BM of both young (i.e., 2-4 months old) mice and aged (i.e., >18 months old) mice using multiple complimentary approaches. We initially utilized a small 0.6 mm combined glass pH electrode to measure pH directly in the bone cavity in vivo (
We incubated cells enriched for hematopoietic stem cells (HSCs) and progenitor cells (LIN-cells) from both young (i.e., 2-4 months old) mice and aged (i.e., >18 months old) mice for 48 hours in a pH ranging from 6.9 to 8.0 (
To determine some effects of changes in pH on HSC function, isolated HSCs (Lin−Sca-1+c-Kit+CD34−Flt3− cells) from BM were exposed to conditions ranging from pH 6.4-7.8 for 48 hours and subsequently competitively transplanted into the bone marrow of lethally irradiated recipients (
To determine cellular mechanisms linked to the enhanced reconstitution potential of HSCs exposed to a pH of 6.9, we next investigated the effect of pH on HSC cell size (
HSCs at pH 6.9 showed less intracellular Reactive Oxygen Species (ROS) (measured via DCF fluorescence) compared to HSC at pH 7.4 in both young and aged HSC, while there appeared to be no difference in ROS in young vs. aged HSCs at the pH 7.4 (
HSCs can exhibit a unique cell metabolism profile. Due to their relative hypoxia, Hif1a is stabilized post-translationally which results in utilization of primarily glycolysis for ATP generation. We next tested whether Hif1a is stabilized in HSCs at a pH 6.9 compared to a pH 7.4. The expression of the Hif1a targets (pdk-1 and vegfa) (
We determined the effect of pH on the structure of the mitochondria in HSCs. Interestingly, we observed elongated and relaxed mitochondria in aged HSCs in contrast to young HSCs that exhibit condensed mitochondria (
Extracellular pH (pHe) can affect intracellular pH (pHi) in multiple ways. We determined the intracellular pH using carboxy SNARF-1 as a fluorescent intracellular pH probe. pH values of HSCs were calculated based on a pH-SNARF-1 standard curve (
Change in cell size as well as volume of HSCs at pHs 6.9 and 7.4 were determined by flow-cytometric and microscopic analyses. Young and aged HSCs presented at pH 6.9 with a decrease in cell size indicated by a decrease in the forward scatter area, which correlates with cell size (
Low pH has been reported to cause a global decrease in HeLa cells and redistribution of the acetylated form of Histone 4 on lysine 16 (H4K16ac) throughout the genome, independently of a specific carbon source or extracellular Na+, Cl−, Ca2+ or phosphate, and it is distinct from changes in the redistribution of H4K16ac in response to nutrient availability. Changes in H4K16ac position within the nucleus of HSCs are also reported to be linked to aging and functional rejuvenation of HSCs. Epigenetic alterations might also be responsible for the “memory” effect of improved function in response to a pH of 6.9 of especially aged HSCs. We thus tested whether changes in pH regulate the distribution of H4K16ac in HSCs. We observed a decrease in the level of H4K16ac at pH 6.9 compared to pH 7.4 in both young and aged HSC using FACS analysis (
We next performed RNA-seq analysis of young and aged HSC at pH 6.9 or 7.4 (
The data reveal extracellular pH as a regulator for stem cell (e.g., murine HSC and human HSC) function and reconstitution ability. A pH of 6.9 in the medium leads to a higher reconstitution ability. An extracellular pH of 6.9 ex vivo also results in at least partial functional rejuvenation of aged murine stem cells (e.g., HSCs). pH regulates stem cell (e.g., HSC) expansion, cellular metabolism and epigenetic polarity. Low cellular oxidation, stabilized metabolism and altered epigenetics induced by a pH of 6.9 are linked to improved stem cell (e.g., HSC) function. These data imply that a pH of 6.9 instead of the standard of 7.4 will support protocols for stem cell (e.g., HSC) maintenance and/or expansion or rejuvenation ex vivo.
The headings used in the disclosure are not meant to suggest that all disclosure relating to the heading is found within the section that starts with that heading. Disclosure for any subject may be found throughout the specification.
It is noted that terms like “preferably,” “commonly,” and “typically” are not used herein to limit the scope of the claimed invention or to imply that certain features are critical, essential, or even important to the structure or function of the claimed invention. Rather, these terms are merely intended to highlight alternative or additional features that may or may not be utilized in a particular embodiment of the present invention.
As used in the disclosure, “a” or “an” means one or more than one, unless otherwise specified. As used in the claims, when used in conjunction with the word “comprising” the words “a” or “an” means one or more than one, unless otherwise specified. As used in the disclosure or claims, “another” means at least a second or more, unless otherwise specified. As used in the disclosure, the phrases “such as”, “for example”, and “e.g.” mean “for example, but not limited to” in that the list following the term (“such as”, “for example”, or “e.g.”) provides some examples but the list is not necessarily a fully inclusive list. The word “comprising” means that the items following the word “comprising” may include additional unrecited elements or steps; that is, “comprising” does not exclude additional unrecited steps or elements.
Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently-disclosed subject matter.
As used herein, the term “about” when referring to a value or to an amount of mass, weight, time, volume, concentration or percentage is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed method.
Detailed descriptions of one or more embodiments are provided herein. It is to be understood, however, that the present invention may be embodied in various forms. Therefore, specific details disclosed herein (even if designated as preferred or advantageous) are not to be interpreted as limiting, but rather are to be used as an illustrative basis for the claims and as a representative basis for teaching one skilled in the art to employ the present invention in any appropriate manner. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
This application claims the benefit of U.S. Provisional Application No. 62/373,015, filed Aug. 10, 2016, entitled “MEDIA WITH A PH TO EXTEND THE LIFE OF STEM CELLS EX VIVO” which is herein incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 62373015 | Aug 2016 | US |
