Neuro-Muscular Differentiation of Adult Porcine Skin Derived Stem Cell-Like Cells

BACKGROUND: Due to the genetic relationship to humans, porcine stem cells are a very important model system to investigate cell differentiation, associated cell signaling pathways, and cell fate. Porcine skin derived stem cells have been isolated from mid-gestation porcine fetus recently. To our knowledge, stem cells from the skin of the adult porcine organism have not been isolated until now. Hence, to our knowledge, we here describe the isolation, expansion, characterization and differentiation of multipotent porcine skin derived stem cell-like cells (pSSCs) from the adult porcine organism for the first time. METHODOLOGY/PRINCIPAL FINDINGS: pSSCs had a spindle shaped morphology similar to mesenchymal stem cells (MSCs). They could be maintained proliferatively active in vitro for more than 120 days and were able to form colonies from single cells. pSSCs expressed Sox2 and Oct3/4, both transcription factors essential to the pluripotent and self-renewing phenotypes of embryonic stem cells, which recently gained attention due to their function in inducing pluripotent stem cells. Furthermore, the expression of the progenitor marker nestin, the somatic stem cell markers Bcrp1/ABCG2, Bmi1, and Stat3 was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in undifferentiated pSSCs. Flow cytometry revealed the expression of the MSC related proteins CD9, CD29, CD44 and CD105, but not CD90. After neuronal differentiation cells with a characteristic morphology of neuronal and smooth muscle-like cells were present in the cultures. Subsequent immunochemistry and flow cytometry revealed the down-regulation of nestin and the up-regulation of the neuron specific protein beta-III-tubulin and the astrocyte marker GFAP. Also, alpha-SMA expressing cells increased during differentiation suggesting the neuro-muscular differentiation of these skin derived cells. pSSCs could also be induced to differentiate into adipocyte-like cells when cultured under specific conditions. CONCLUSIONS/SIGNIFICANCE: Adult porcine skin harbors a population of stem cell-like cells (pSSCs) that can be isolated via enzymatic digestion. These pSSCs show characteristic features of MSCs originated in other tissues and express the embryonic stem cell marker Oct3/4, Sox2, and Stat3. Furthermore, pSSCs have the potential to differentiate into cells from two different germ lines, the ectoderm (neurons, astrocytes) and the mesoderm (smooth muscle cells, adipocytes)

What is required to combine human biomonitoring and health surveys?

© 2022 The Author(s). Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).Obtaining holistic information about health and health determinants at the population level should also include data on environmental risk factors of health. So far, only a few countries have combined, at the national level, health and human biomonitoring (HBM) surveys to collect extensive information on health, lifestyles, biological health determinants and environmental exposures. This paper will provide guidelines on how to combine health and HBM surveys and what is the added value of doing so. Health and HBM surveys utilize similar infrastructure and data collection methods including questionnaires, collection and analysis of biological samples, and objective health measurements. There are many overlapping or comparable steps in these two survey types. At the European level, detailed protocols for conducting a health examination survey or HBM study exists separately but there is no protocol for a combined survey available by now. Our recommendations for combined health and HBM surveys focus on a cross-sectional survey on general population aged 6-79 years. To avoid unnecessary participant burden, for the selection of included measurements basic principle would be to ensure that results of the measurements have a public health relevance and clear interpretation. Combining health and HBM surveys into one survey would produce an extensive database for research to support policy decisions in many fields such as public health and chemical regulations. Combined surveys are cost-effective as only one infrastructure is needed to collect information and recruit participants.This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 733032.info:eu-repo/semantics/publishedVersio

Discovering time-trends of the German populations exposure to contaminants by analysis of human samples of the German Environmental Specimen Bank (ESB)

The German Environmental Specimen Bank (ESB) is a monitoring instrument of the German Federal Ministry for the Environment, Nature Conservationand Nuclear Safety. The permanent biobank facility is run since 1981 containing environmental and human samples from Germany. All samples are collected according to standard operating procedures (SOP). An annually standardized collection of human samples at four different regional sites of the country has been established since 1997. Routine sampling is done once a year, recruiting healthy non occupationally exposed students aged 20-29 years, in an equal gender distribution. The number of participants recruited is approximately 120 students per site and year. Directly after the annual sampling process, the human samples are analyzed for selected environmental chemicals. The time-trends of lead in blood, mercury and pentachlorophenol in 24 h-urine and polychlorinated biphenyls in plasma demonstrated a decrease of exposure during the last two decades by about 40 – 90 percent. In parallel retrospective studies using cryo-archived samples revealed increasing time trends of emerging chemicals used as substitutes for regulated toxicants. The data demonstrates the great relevance of the ESB for the health related environmental monitoring and shows the importance of human biomonitoring as a tool in information based policy making

Assessing background contamination of sample tubes used in human biomonitoring by non-targeted liquid chromatography–high resolution mass spectrometry

Controlling and minimising background contamination is crucial for maintaining a high quality of samples in human biomonitoring targeting organic chemicals. We assessed the contamination of three previous types and one newly introduced medical-grade type of sample tubes used for storing human body fluids at the German Environmental Specimen Bank. Aqueous extracts from these tubes were analysed by non-targeted liquid chromatography-high resolution mass spectrometry (LC-HRMS) before and after a dedicated cleaning procedure. After peak detection using MZmine, Bayesian hypothesis testing was used to group peaks into those originating either from instrumental and laboratory background contamination, or actual tube contaminants, based on if their peak height was reduced, increased or not affected by the cleaning procedure. For all four tube types 80–90% of the 2475 peaks (1549 in positive and 926 in negative mode) were assigned to laboratory/instrumental background, which we have to consider as potential sample tube contaminants. Among the tube contaminants, results suggest a considerable difference in the contaminant peak inventory and the absolute level of contamination among the different sample tube types. The cleaning procedure did not affect the largest fraction of peaks (50–70%). For the medical grade tubes, the removal of contaminants by the cleaning procedure was strongest compared to the previous tubes, but in all cases a small fraction increased in intensity after cleaning, probably due to a release of oligomers or additives. The identified laboratory background contaminants were mainly semi-volatile polymer additives such as phthalates and phosphate esters. A few compounds could be assigned solely as tube-specific contaminants, such as N,N-dibutylformamide and several constituents of the oligomeric light stabiliser Tinuvin-622. A cleaning procedure before use is an effective way to standardise the used sample tubes and minimises the background contamination, and therefore increases sample quality and therewith analytical results

Expression of stem cell related genes and translated proteins.

<p>(A) RT-PCR analysis of stem cell related genes. Line 1–3: results of three independent cell strains, line 4: negative control (-RT) from one representative cell strain. (B) Flow cytometric analysis of cell surface markers CD9, CD29, CD44, CD90, CD105, and nestin. Data are expressed as means ± SEM from three independent pSSCs strains after 22 CPD. (C) Immunocytochemistry revealed a subpopulation of pSSCs expressing nestin. (D) Double staining indicates the coexpression of nestin and fibronectin. Scale bars: 50 µm.</p

Induced adipogenic differentiation of pSSCs.

<p>(A) The adipogenic lineage differentiation is shown by bright field pictures of positive Oil Red O stained lipid vacuoles within the cytoplasm of the pSSCs after 30 days of induced differentiation. (B) Oil red O staining of pSSCs cultured in control medium. Scale bars: 50 µm. (C) Leptin expression shown by RT-PCR in adipogenic differentiated and undifferentiated pSSC cultures. Hypoxanthin-guanin phosphoribosyltransferase (HPRT) was used as an internal control for each PCR.</p

Trends in characteristics of 24-h urine samples and their relevance for human biomonitoring studies: 20 years of experience in the German Environmental Specimen Bank

To document trends in human exposure to environmental pollutants, the German Environmental Specimen Bank (ESB) has been routinely collecting and archiving 24-h urine samples from young adults at four sampling sites in Germany on an annual basis. For the purpose of normalizing measured analyte concentrations, urinary creatinine (UC), specific gravity (SG), conductivity (CON), and total urine volume (UVtot) of 24-h urine samples have also been recorded. These parameters are however susceptible to variation over time, as well as within/among participants and normalization against them can thus affect the interpretation of data regarding exposure to environmental pollutants. To evaluate the influence of normalization against these parameters, we first sought to determine variations of these parameters with regard to differences between sexes and trends over time. We analysed data from 8619 urine samples collected from 1997 to 2016. We observed an inverse relation between UVtot and UC, SG, and CON. We also found differences between sexes for UC, SG and CON, but not UVtot. UC, SG, and CON showed significant decreasing trends over time in both sexes. In contrast, a significant increase of over 30% in UVtot, independent of participant age and BMI, was revealed. This increase in UVtot and the concomitant sample dilution is likely to have an impact on measured analyte concentrations in 24-h urine samples. Hence, normalization of urinary concentrations is warranted when interpreting time trends of human exposure. Next, urinary calcium (Ca2+) concentrations of ESB participants were used to demonstrate the effects of normalization against each of the four urine parameters. From 1997 to 2016, measured Ca2+ concentrations showed a statistically significant but scientifically implausible decrease. Normalization of Ca2+ concentrations against UVtot (by calculating the total daily excretion), UC, or CON, but not SG, eliminated this decrease. Consistent with previous work, Ca2+ concentrations in urine and total daily Ca2+ excretion were higher for males than females. Normalization against UC, SG, or CON, however, attenuated this difference. Thus, to avoid misinterpretation in trend analysis and sex-specific excretion in 24-h urine samples, the calculation of the total daily excretion is recommended

Growth characteristics of pSSCs in vitro.

<p>(A) Proliferation of pSSCs: Given is the CPD (cumulative population doubling) of pSSCs as function of days in culture. Data are expressed as means ± SEM from three independent pSSC strains. (B) Average CFU efficiency of one representative pSSCs strain after 22 CPD. Mean of three experiments ± SEM. (C) Image of cell colonies (stained with trypan-blue) formed after 11 days seeded with 10 cells per cm² growth surface. Scale bar: 1 cm. (D, E) Morphological appearance of pSSCs. Scale bars: (D) 200 µm, (E) 100 µm.</p

Neuro-muscular differentiation potential of pSSCs.

<p>(A) After 30 days of induced neuronal differentiation one cell strain developed a structured cell network of linked bodies. (B–D) Cells show morphologies similar of neuronal cells (white arrows) and smooth muscle cells (black arrows). Scale bars: (A, B) 500 µm, (C) 50 µm, (D) 100 µm. (C) Represents the marked region in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008968#pone-0008968-g003" target="_blank">Fig. 3B</a>. (E–G) Immunocytochemistry revealed the expression β-III-tubulin and GFAP in the centre and some surrounding cells of the bodies, whereas α-SMA is exclusively expressed in the outer regions of the bodies and some surrounding cells. Scale bars: 50 µm. (H, I) β-III-tubulin is expressed in a subpopulation of cells that are morphological consistent with neurons. (J, K) Other subpopulations of differentiated pSSCs expressed the neuronal markers GFAP and neurofilament M. (L, M) Subpopulations with a morphology consistent of smooth muscle cells expressed the smooth muscle marker α-SMA. Scale bars: 50 µm.</p