Much of the work conducted on adult stem cells has focused on mesenchymal stem cells (MSCs) found within the bone marrow stroma. Adipose tissue, like bone marrow, is derived from the embryonic mesenchyme and contains a stroma that is easily isolated. Preliminary studies have recently identified a putative stem cell population within the adipose stromal compartment. This cell population, termed processed lipoaspirate (PLA) cells, can be isolated from human lipoaspirates and, like MSCs, differentiate toward the osteogenic, adipogenic, myogenic, and chondrogenic lineages. To confirm whether adipose tissue contains stem cells, the PLA population and multiple clonal isolates were analyzed using several molecular and biochemical approaches. PLA cells expressed multiple CD marker antigens similar to those observed on MSCs. Mesodermal lineage induction of PLA cells and clones resulted in the expression of multiple lineage-specific genes and proteins. Furthermore, biochemical analysis also confirmed lineage-specific activity. In addition to mesodermal capacity, PLA cells and clones differentiated into putative neurogenic cells, exhibiting a neuronal-like morphology and expressing several proteins consistent with the neuronal phenotype. Finally, PLA cells exhibited unique characteristics distinct from those seen in MSCs, including differences in CD marker profile and gene expression.

/pdf/human-adipose-tissue-is-a-source-of-multipotent-stem-cells-18jo72s52v.pdf

Human Adipose Tissue Is a Source of Multipotent Stem Cells

With few exceptions biological tissues strongly scatter light, making high-resolution deep imaging impossible for traditional⎯including confocal⎯fluorescence microscopy. Nonlinear optical microscopy, in particular two photon–excited fluorescence microscopy, has overcome this limitation, providing large depth penetration mainly because even multiply scattered signal photons can be assigned to their origin as the result of localized nonlinear signal generation. Two-photon microscopy thus allows cellular imaging several hundred microns deep in various organs of living animals. Here we review fundamental concepts of nonlinear microscopy and discuss conditions relevant for achieving large imaging depths in intact tissue.

/pdf/deep-tissue-two-photon-microscopy-2aqu6d6n65.pdf

Deep tissue two-photon microscopy

Multiphoton microscopy (MPM) has found a niche in the world of biological imaging as the best noninvasive means of fluorescence microscopy in tissue explants and living animals. Coupled with transgenic mouse models of disease and 'smart' genetically encoded fluorescent indicators, its use is now increasing exponentially. Properly applied, it is capable of measuring calcium transients 500 microm deep in a mouse brain, or quantifying blood flow by imaging shadows of blood cells as they race through capillaries. With the multitude of possibilities afforded by variations of nonlinear optics and localized photochemistry, it is possible to image collagen fibrils directly within tissue through nonlinear scattering, or release caged compounds in sub-femtoliter volumes.

/pdf/nonlinear-magic-multiphoton-microscopy-in-the-biosciences-3fszv5aq8z.pdf

Nonlinear magic: multiphoton microscopy in the biosciences

Observed atmospheric concentrations of CO2 and data on the partial pressures of CO2 in surface ocean waters are combined to identify globally significant sources and sinks of CO2. The atmospheric data are compared with boundary layer concentrations calculated with the transport fields generated by a general circulation model (GCM) for specified source-sink distributions. In the model the observed north-south atmospheric concentration gradient can be maintained only if sinks for CO2 are greater in the Northern than in the Southern Hemisphere. The observed differences between the partial pressure of CO2 in the surface waters of the Northern Hemisphere and the atmosphere are too small for the oceans to be the major sink of fossil fuel CO2. Therefore, a large amount of the CO2 is apparently absorbed on the continents by terrestrial ecosystems.

https://science.sciencemag.org/content/sci/247/4949/1431.full.pdf

Observational contrains on the global atmospheric co2 budget.

Capacitation of bovine sperm was evaluated by determining the ability of sperm to fertilize bovine oocytes in vitro and to undergo an acrosome reaction upon exposure to lysophosphatidylcholine (LC). Incubation of sperm with heparin (10 micrograms/ml) increased the percentage of oocytes fertilized, but this required exposing sperm to heparin for at least 4 h before adding them to oocytes. There was no effect on the percentage of motile or acrosome-reacted sperm after exposure of noncapacitated sperm to 100 micrograms/ml LC for 15 min. When sperm were incubated for 4 h with heparin, exposure to 100 micrograms/ml LC for 15 min had no effect on the percentage of sperm that were motile, but the percentage of acrosome-reacted sperm increased from less than 10% to over 70%. The acrosome reactions (ARs) induced by LC were synchronous, reached maximal levels within 15 min, and differed (p less than 0.001) between sperm incubated under capacitating (with heparin) and noncapacitating conditions (without heparin). The time course required for heparin to capacitate sperm as judged by in vitro fertilization and to render sperm sensitive to LC induction of the AR were found to be similar. The percentage of ARs induced by LC and percentage of oocytes fertilized by sperm were found to be heparin-dose-dependent, with the maximum responses occurring at 5-10 micrograms/ml heparin. The correlation between the mean fertilization and LC-induced AR percentages was 0.997 (p less than 0.01). These studies demonstrate capacitation of bovine sperm by heparin requires at least a 4-h exposure of sperm to heparin and suggest that plasma membrane changes prior to an AR can be detected by exposure of bovine sperm to LC.

Capacitation of bovine sperm by heparin.

One-cell CF-1 x B6SJLF1/J embryos, which usually exhibit a 2-cell block to development in vitro, have been cultured to the blastocyst stage using CZB medium and a glucose washing procedure. CZB medium is a further modification of modified BMOC-2 containing an increased lactate/pyruvate ratio of 116, 1 mM-glutamine and 0.1 mM-EDTA but lacking glucose. Continuous culture of one-cell embryos in CZB medium allowed 83% of embryos to develop beyond the 2-cell stage of which 63% were morulae at 72 h of culture, but blastocysts did not develop. However, washing embryos into CZB medium containing glucose after 48 h of culture (3-4-cell stage) was sufficient to allow development to proceed, with 48% of embryos reaching the blastocyst stage by 96 h of culture. Exposure of embryos to glucose was only necessary from the 3-4-cell stage through the early morula stage since washing back into medium CZB without glucose at 72 h of culture still promoted the development of 50% of embryos to the blastocyst stage. The presence of glucose in this medium for the first 48 h of culture (1-cell to 4-cell stage) was detrimental to embryo development. Glutamine, however, exerted a beneficial effect on embryo development from the 1-cell to the 4-cell stage although its presence was not required for development to proceed during the final 48 h of culture. Blastocysts which developed under optimum conditions contained an average of 33.7 total cells. The in-vitro development of 1-cell embryos beyond the 2-cell stage in response to the removal of glucose and the addition of glutamine to the culture medium suggests that glucose may block some essential metabolic process, and that glutamine may be a preferred energy substrate during early development for these mouse embryos.

/pdf/an-improved-culture-medium-supports-development-of-random-4ommimicna.pdf

An improved culture medium supports development of random-bred 1-cell mouse embryos in vitro

Mammalian preimplantation embryos normally develop within the protected environment of the female reproductive tract, which virtually precludes studies on embryogenesis in situ. Information must therefore be derived from experiments on cultured embryos. Consequently, studies on the epigenetic regulation of embryogenesis have long been interwoven with efforts to formulate culture media capable of sustaining normal development. In this review, comparative information on epigenetic regulation of embryo development is discussed, including information on energy substrate and amino acid preferences of embryos. Advantages of simple versus complex culture media, and of substituting serum albumin or synthetic macromolecules for serum, are discussed. Some potential pitfalls of co-culture are described. Culture appears to induce anomalies in embryo metabolism, which may derive from disturbed intracellular pH. Rationales for selecting endpoints to evaluate the outcome of experiments are considered, including incorporation of timing of embryo development into the analysis. Poor experimental design and/or data analysis can detract from or even negate the value of data obtained from embryo culture; examples are examined to help correct this problem. All of these points are discussed with a view to using data on the needs of embryos for making improvements in the design of culture media, so that higher yields and increased viability of embryos are achieved.

Culture of preimplantation embryos: facts and artifacts

Eight-cell embryos were recovered from mated golden hamsters that had been superovulated with pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). Embryos were cultured for 24 or 32 h in a defined medium (modified Tyrode's solution) designed for fertilization of hamster oocytes in vitro. This medium was supplemented in some experiments with amino acids (glutamine, phenylalanine, methionine and isoleucine) and with vitamins (Eagle's Minimum Essential Medium vitamin supplement). At the end of the culture period, the numbers of embryos developing to the blastocyst stage were recorded. In other experiments, the effects of varying the osmotic pressure (225, 250, 275 and 300 m0smol/kg) and the pH (6.8 and 7.4) of the culture medium on blastocyst formation were examined. A difference was found between the ability of early 8-cell embryos (approx. 54 h post-egg activation) and late 8-cell embryos (approx. 62 h post-egg activation) to develop in culture. In the unsupplemented culture medium, only 2% of early 8-cell embryos developed to the blastocyst stage compared with 22% of late 8-cell embryos. A marked effect of the four amino acids on development was found. In the presence of amino acids 36% of early 8-cell embryos developed into blastocysts (18-fold increase). The amino acids also increased the percentage of late 8-cell embryos that developed into blastocysts from 22% to 66%. These data suggest that an important metabolic change may occur in hamster embryos during a critical period at the 8-cell stage of development. No additional effect on development was observed when vitamins were included in the culture medium. No significant effect of either osmotic pressure of pH of the culture medium on development was found. When blastocysts formed from cultured 8-cell embryos were transferred surgically to pseudopregnant hamsters, about 25% developed into normal-looking fetuses and 5 normal-looking young were born, 4 of which have survived. These results represent an approach towards achieving complete preimplantation development of hamster embryos in vitro.

Development of preimplantation embryos of the golden hamster in a defined culture medium.

A major challenge for fluorescence imaging of living mammalian cells is maintaining viability following prolonged exposure to excitation illumination. We have monitored the dynamics of mitochondrial distribution in hamster embryos at frequent intervals over 24 h using two-photon microscopy (1,047 nm) while maintaining blastocyst, and even fetal, developmental competence. In contrast, confocal imaging for only 8 h inhibits development, even without fluorophore excitation. Photo-induced production of H2O2 may account, in part, for this inhibition. Thus, two-photon microscopy, but not confocal microscopy, has permitted long-term fluorescence observations of the dynamics of three-dimensional cytoarchitecture in highly photosensitive specimens such as mammalian embryos.

Barry D. Bavister

Papers

An improved culture medium supports development of random-bred 1-cell mouse embryos in vitro

Culture of preimplantation embryos: facts and artifacts

Development of preimplantation embryos of the golden hamster in a defined culture medium.

Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability

Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability