Stem Cell Biology

Stem Cell Biology

  • Naina Bhatia-Dey added an answer:
    What is the OCT4 expression pattern in early embryo during gastrulation, especially in the mesoderm?

    I am working on mesodermal differentiation of embryonic stem cells. I was wondering if OCT4 expression is still maintained during gastrulation in the embryonic disk? Moreover, is the early embryonic mesodermal cell (when they start to express brachyury) OCT4 positive? Thanks in advance for your replies.

    Naina Bhatia-Dey · Howard University

    A 2013 study in Nature Cell Biology indicates that Oct-4 expression is required for differentiation in all three cell lineages in vitro, however, in-vivo (in embryos as well as cell-lines) it could be different.

    In current research climate where trace amount of transcript can be detected by sophisticated technology, it is difficult to predict if a trace amount would be found in certain mutants or specific cell lines

  • Glen Lester Sequiera added an answer:
    Has anyone used Matrigel instead of Vitronectin for reprogramming with Sendai virus?

    I will reprogram human fibroblasts and CD34+ HSC with Sendai virus (CytoTune®-iPS 2.0). In the protocol they recommend coating with the plates with vitronectin, but as far as I know vitronectin is an alternative to matrigel. I already have matrigel, and wonder if it would make a difference in reprogramming efficiency. Has anyone tested it?

    Glen Lester Sequiera · University of Manitoba

    Hi Ceren. As the 3 of our fellow researchers have mentioned before me, Matrigel is not a problem. I was part of something like release candidate evaluation for the kit. There were no major changes when I carried out reprogramming for around 25 different lines of fibroblasts. The main thing here is the medium. E6 or E8 is important. I tried mTeSR and StemPro but its efficacy was low. Hope this helps. Have a good day.

  • María I Vaccaro added an answer:
    Has anyone worked on measuring autophagy induced genes by realtime PCR?

    I am trying to see the autophagy levels by inducing with the Rapamycin  in a mammalian cells and measure the gene levels by Real time PCR.  Has anyone worked with this concept? I need your suggestions?

    María I Vaccaro · University of Buenos Aires

    VMP1 is a new autophagy related gene, which expression triggers autophagy in mammalian cells. We use QPCR of VMP1 to know if VMP1-mediated autophagy is activated.

    J Biol Chem. 2007 Dec 21;282(51):37124-33 

  • Carlos Bueno-Betí added an answer:
    How can I achieve Endothelial Progenitor Cell Isolation - CD45+ cells in culture?
    We are trying to isolate endothelial progenitor cells (EPC) from peripheral blood. We tried with densitiy gradient centrifugation and CD34+ selection of resulting PBMC with EasySep. Cells are subsequently cultured in endothelial growth media on fibronectin coated plates. However, after CD34 positive isolation there are almost no cells left! Although we stick to the manual.
    If we leave out CD34 positive selection and plate PBMC directly in endothelial medium, we get adherent cells which are CD45+ and vWF+, but VEGFR-, CD34- and CD31-. What kind of cells is this? Why are there no endothelial cells in the culture?
    We already varied the anticoagulant, endothelial media with and without FCS, different Easysep cocktail volumes, harder centrifugation, and buffers (PBS2-, Hanks).
    Carlos Bueno-Betí · Fundación de Investigación del Hospital Clínico Universitario de Valencia INCLIVA

    The cell you obtained are likely to be early EPC or circulating angiogenic cells (CAC).

    These cells appear very early during the culture (within the first 7 days). They also express CD14 (monocle marker) and are believe to promote vasculogenesis by acting in a paracrine way. They have low proliferative potencial in vitro and the do not form tube-like structures on Matrigel matrix.

    If you want to obtain endothelial cells in culture you will have to wait for them to appear at least 13 days.  They show the typical cobble-stone morphology of EC in culture, have a high proliferative capacity and they form in vitro tube-like structures on Matrigel. Last year we published this papers  "An affordable method to obtain cultured endothelial cells from peripheral blood". Here we propose an standardized protocol for EPC isolation and culture from peripheral blood samples. Isolated EPC by using this protocol showed EC-like morphology, phenotype and function. Please read it, it may help you.

    Carlos Bueno-Betí

  • Syed Raza added an answer:
    How can I successfully transduct mouse adipose tissue-derived mesenchymal stem cells lentivirus ?

    I want to transduct mouse Adipose Tissue-Derived Mesenchymal Stem Cells with lentiviral that expression GFP buttransduction don't succeed, GFP expression is not detectable .
    I use working concentration for polybrene is 8 to 10 μg/ ml.microgram/ml) for transduced For 12 to 14 hours.

    Syed Raza · University of Utah

    Hi Hori you should try RetroNectin

    Best of Luck

  • Bipasha Bose added an answer:
    How can I store rats organs for future (stem) cell extraction?

    I currently want to dissect a rat and store its organs inside liquid nitrogen for future cells or stem cell extraction, including heart, liver, kidney, bone marrow, tendon, cardiomuscle, etc. I have questions:

    1) What kind of media should I use?

    2) Can I store them in -86 C Freezer instead of LN2 for long periods of time (few months)?

    3) What is the precaution I should notice during the thawing process?

    Thanks a lot for your answers,


    Long.

    Bipasha Bose · Nanyang Technological University

    Isolation of the stem cells from fresh tissues is always desirable. However, liquid nitrogen storage of the tissue is an alternative. 

  • Daniel Seidel added an answer:
    Why are DC cells derived from murine BmDCs not stimulated in vitro with CpG?

    I differentiated DC cells from murine BmDCs to test the adjuvanticity of a protein using CpG as a positive control. When quantifying cytokines, no immune response was detected in non stimulated cells, stimulated cells with the protein and stimulated cells with CpG. Does anyone have any explanation for why DC cells are not stimulated even in the positive control? Could the differentiation protocol I used be causing this?

    Thank you

  • Aman Chowdhry added an answer:
    What may the best method be to isolate dental pulp from tooth to extract stem cells?
    I wish to work on mesenchymal dental pulp stem cells.. I need to know the best procedure so that the viability of stem cells is maintained when extracted from dental pulp.
    Aman Chowdhry · Jamia Millia Islamia

    kindly work instead of wishing only..

    (i know it require hell lot of ethical clearance)

    all the very best

  • Elena Yu. Filinova added an answer:
    Can I loose the protein of interest from a stable clone?

    hi,

    Iam facing a weird problem after transfection. I am using C3H10 meschencymal cell line which is a stable clone expressing a protein which is GFP tagged. Iam using this cell line to transfect another protein having a red tag. both the protein has G418 resistance. After a month, I have few colonies. But when I see it under the fluorescent microscope the resistant cells neither have GFP or red tag.

    1 . How is this possible,  when I have used a stable clone with a GFP protein ( I did check the cell for fluorescence before the experiment.) for transfection.

    2. Do the cells loose the protein for any reason.

    Suggestions are welcome

    Elena Yu. Filinova · Advanced Biomedical Research Laboratory, RF; Vidipharm Gmbh, Austria

    If this happened you can only transfect/infect cells newly, make colonies selection and produce new stably transfected line. Double-transfectants are even less stable. After several years of exercising with different vectors and attempts to obtain stable transfectants (not unsuccessful but not efficient enough for in vivo works) I started to make results in vitro within 3 weeks of first experession when it is combined transient-genomic and high enough. Mr. Reddy's advise to freeze stable transfectants before expression is lost is very useful.

    Greetings

  • Andrey Luchnik added an answer:
    How is immortal strand hypothesis acceptable?

    During replication of DNA scientist shown that stemm cell retain their own copy of DNA and avoid mutation in genome ,but how it can be possible ,and is it possible in other cell type

    Andrey Luchnik · Russian Academy of Sciences

    Yes, indeed, dear William. But because these sequences are tandem repeats it is often difficult to follow their shortening. One example is telomeric DNA shortening. Other examples are cited very rarely. 

    But these shortenings are extremely important for gene switching during determination of cell lineages. This mechanism is considered in my paper "DNA conformational transitions induced by supercoiling control transcription in chromatin" (2014).

  • Dirk Breitkreutz added an answer:
    Can anyone suggest a non-invasive procedure to obtain fibroblasts from patients?

    We will need primary cells from patients that have a rare inherited metabolic disease. We will be turning them in to IPS and then differenciate them into hepatocytes. We have to use a non-invasive method to obtain primary cells and right now we are planning isolate keratinocytes from hair bulbs following Aasen &  Belmonte's procedure ( http://www.nature.com/nprot/journal/v5/n2/full/nprot.2009.241.html ) but since working with keratinocytes is somewhat more tedious and expensive than working with fibroblasts, we would like to be able to isolate fibroblasts via a non-invasive method. 

    Can anyone suggest such method or alternative? 

    Also would it be possible to obtain fibroblast culture from hair bulbs? 

    Regards.

    Dirk Breitkreutz · Hochschule Mannheim

    As Dorothy M Supp is suggeting small punch biopsies should be feasible to isolate enough skin fibroblasts for growth in vitro, though this is of course an (minimally) invasive procedure. On the other hand, when you properly plug hair follicles, thus maintaining the hair bulb you will get also the dermal papilla. This is a condensation of specialized fibroblast-like stromal cells required for renewal of the hair cycle (see e.g.  older papers of Alain Limat et al.). In culture these cells may behave like skin fibroblasts.

  • Mohammad Esmaeillou added an answer:
    Why do cancer stem cells sometimes exhibit high proliferation in vitro but exhibit less proliferation when placed in vivo in a tumor condition?

    When i have checked the proliferation marker (Cyclin D1) in cancer stem cell (CSC) then its expression was higher. However when i have developed the in vivo tumor which is highly aggressive and re-checked the status of cyclin D1 in these total tumors lysate by western. Result showed that there were no change of cyclin D1 expression in CSC in contrast to non-CSC, irrespective of tumors volume.

    Mohammad Esmaeillou · The University of Calgary

    so thank u for ur suggestionsss

  • Tapan Mukherjee added an answer:
    How can I measure HUVEC express P-selectin and a derivative/P-selectin interaction?

    I'm starting to work with HUVEC and several works do this assay with HUVEC, but the majority induces in different manners.

    Tapan Mukherjee · Maharishi Markandeshwar University, Mullana

    I have worked with several adhesion molecules like VCAM-1, ICAM-1, RAGE and selectins. The best way to check them is by PCR/real time PCR since proteins level of some of them (VCAM-1/selectins) in HUVEC is too low to detect by Western blot /immunohistochemistry/ELISA etc. I have stimulated HUVEC with TNF-alpha to induce the level of VCAM-1/p-selectin  proteins. If Western blot does not work immunocytochemistry/immunohistochemistry may be used.

  • Sher Bahadur Poudel added an answer:
    What is appropriate media for MSCs culture?

    I would like to know using Alpha MEM having ascorbic acid is good idea or not for mesenchymal stem cells culture. I generally use mice/rat bone marrow stromal cells for MSCs. using alpha MEM containing ascorbic acid induces differentiation of MSCs. I would be happy to know which media is best for MSCs culture.

    Sher Bahadur Poudel · Chonbuk National University

    Thank you very much....

  • Inese Cakstina added an answer:
    Stemness marker for human bone marrow derived MSC - can anyone help?
    Currently so many people are working with mesenchymal stem cells and they use surface markers for the validation of cells to be positive for CD90, CD73, and negative for CD45 and other kind of markers. But with none of these markers you can be really sure that you have real MSC WITH STEMNESS QUALIFICATIONS. There are so many articles and people use a huge set of markers to prove they have MSC and it is also very difficult to dig up the right information because of a huge number of articles. I want to know based of your experiences can you suggest a marker which is easy to go? And in other words is there even any marker? I also know about Stro-1 and CD 105 but these Markers are not real marker for stemness.
    Inese Cakstina · Riga Stradins University

    I found this article interesting - sorting MSC's by physical parameters: http://www.ncbi.nlm.nih.gov/pubmed/25411477

    Here is the press release from MIT: newsoffice.mit.edu/2014/identify-rare-stem-cells-in-bone-marrow-1006?elq=1540593920034fc997f3c32c69bf7875&elqCampaignId=4

  • Maurice Canham added an answer:
    Have you seen a difference between E8/Vitronectin and mTESR/Matrigel for iPSC, in terms of differentiation ability or chromosomal stability?

    We have observed several iPSC clones with chromosomal abnormalities grown in E8/Vitronectin and poor neuronal differentiation ability and wonder if this is purely co-incidence or a culture effect.

    Maurice Canham · The University of Edinburgh

    Hi,

    We have noted that the high cntent of FGF2 present in Essential 8 can indeed be inhibitory to differentiation. Seeding cells in a low fgf containing media (like Nutristem) can overcome this. Alternatively, we have tried seeding in E8 but then adding PD03 for one day and this also helped our protocol. I'm not sure about the effects of vitronectin versus matrigel since we are using Laminin 521. In terms of chromosomal abnormalities, do you directly compare karyotpyes (or SNP data) of cultures of  E8/Vitronectin and mTESR/Matrigel at the same passage and find a difference? Abnormalities can increase with passage for hEPCs. See attached papers relating to fgf signalling and self-renewal/differentiation

  • Marin Jukic added an answer:
    Can someone point on a good review/articles describing signaling cascades in colon development in mouse embryo?

    Wnt, Hh, Bmp's etc. Gene-expression/ protein changes marking stages in normal colon development

    Marin Jukic · Karolinska Institutet

    Thanks a lot

  • Islam Saadeldin added an answer:
    What are the factors affecting stem cells differentiation in vivo or in vitro?
    There are several factors affecting stem cells differentiation during embryonic development stage. I would like to discuss these factors together through your research and your experiences. Either in vivo or in vitro .
    Islam Saadeldin · Zagazig University

    From my experience; using feeder cells maintain the normal morphology of oligopotent ESCs (trophoblasts) however using Matrigel (basement membrane matrix) stimulate differentiation into elongated spindle like cells. 

    I would like to share a time-lapse video I captured during my experiment.

    https://www.youtube.com/watch?v=laLia1RVmEc

  • Vahid Razban added an answer:
    Why are mesenchymal stem cells obtained from mouse adipose tissue spread in morphology?

    Why are mesenchymal stem cells obtained from mouse adipose tissue spread in morphology?

    I am culturing Mesenchymal stem cells from mouse adipose tissue (epididymal fat tissue).Fat tissue is minced properly and digested using 0.05% collagenase type 4. The cells are then seeded in T-25 flask in IMDM 10% FBS containing media. Culture media is then changed after 48hrs. Cells trypsinized when 80% confluent using 0.25% trypsin.

    Phenotypic surface marker expression of MSC at P-3 , CD29= 78.1% ,CD73= 21.2% ,CD44= 6.78% ,SCA1= 21.8% ,CD31= 4.22%.

    Is it that MSCs obtained from adipose tissue are spreaded in morphology?

    Do I need to change the culture conditions?

    Vahid Razban · National Institute of Genetic Engineering and Biotechnology

    Murine mesenchymal stem cell isolation differs from human counterparts isolation at least in some parts. Murine MSCs seems to be immediately enter a sleep period during which cells start to die, detach and decrease in number. After several medium replacements (2-3 times/week) they start to proliferate and gain to their spindle shape. Its my experiencesHope to be useful.

  • Yves Gérard Illouz added an answer:
    Do all patients' adipose SVF cells freeze?

    We have noticed that some patients' cells freeze and thaw easily and those patients get good clinical response to frozen cells. There is a group who do not respond well to frozen cells. In the vet world we see some bulls whose semen will not freeze. Is this the same for human SVF cells?

    Yves Gérard Illouz · Ex head of department of plastic surgery at Saint Louis Hospital, Paris, France

    Normally, no problems !

  • Andrew S Brack added an answer:
    Is there evidence for non-satellite cell resident muscle progenitors?
    On ResearchGate, there has been a very collegial discussion about the role of satellite cells during muscle hypertrophy. A number of us also cited increasing evidence that non-satellite cells play a role as niche support cells and/or can directly participate in the myogenic lineage. So, hoping for a fruitful discussion, lets start to put this data together to see if it forms a coherent picture? Jump in.
    Andrew S Brack · Harvard University

    Thanks for clarifying. 

  • Sandeep Rajput added an answer:
    Which marker is best for staining of breast cancer stem cells by immunohistochemistry?

    I have been collecting breast tumor tissues from patients and am going to study the expression profiling of stem cell regulatory genes. Also, i would like to do assessment of breast cancer stem cells in tissue section by immunohistochemistry.

    but am in little bit of confusion regarding markers. Which marker is best for assessment ? ( ALDH, CD133, CD24, CD44) 

    Sandeep Rajput · Washington University in St. Louis

    You can also include ESA (Epithelial specific antigen)

  • Elena M Glinka added an answer:
    What kind of plant nanofibers have positive and negative effects on human stem cells?
    I want to now which plants nanofiber has not genotoxicity effect on human cells

    Hello,

    According to de Lima et al., (Int J Nanomedicine. 2012;7:3555-65) and (Clift et al. (Biomacromolecules. 2011 Oct 10;12(10):3666-73) cellulose nanofibers isolated from cotton did not cause any significant DNA breaks in the cell types employed (lymphocytes, fibroblasts, human lung cells).

    Good luck!

  • Sayandip Mukherjee added an answer:
    Can someone answer the questions below regarding correct culturing procedure for the reprogramming of mouse iPSC ?

    Additional details include multiple questions:

    1) Can mouse iPSC be cultured in feeder free conditions. Kindly provide the details of the media and matrix that could be used for the feeder free culture of mouse iPSC.

    2) Passaging of mouse iPSC can be done using Trysin?? Or other methods including mechanical dissociation/milder enzymes such as collagenase or dispase are required??

    3) Can the somatic cells after protein transduction be directly maintained in feeder free condition with the media supplement of the conditioned iPSC media from mouse MEF's?? Or its is important to grow cells in presence of feeder layer ducring initial reprogramming phase??? 

    Kindly reply

    Regards,

    Tarun

    Sayandip Mukherjee · Bangalore University

    Hi Tarun:

    You will get all your answers in this publication:

    http://www.ijdb.ehu.es/web/paper.php?doi=10.1387/ijdb.130173to

    For humans:

    http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0087151

    Hope that helps!

    Bw,

  • Steingrimur Stefansson added an answer:
    What buffers are safe for in vivo use?
    I read a study (link below) where they cultured cells and re-suspended them in a buffer before injection. I am curious what buffers are safe for this? PBS? aMEM? What do you think?
    Steingrimur Stefansson · HeMemics Biotechnologies Inc

    Hi Ellie, if you are going to inject cells into animals, the best way is to use tissue culture grade media, DMEM, EMEM, whatever.

    These are sterile media that  have minimal endotoxin and other pyrogens in them.

    You just have to be careful about using sterile techniques to prep. the cells for injection. 

  • Constantine Kaniklidis added an answer:
    Anybody knows a good review on HP-1 and its role in cell senescence?

    I am interested in the role of HP-1 in cellular senescence. We know that HP-1 is involved in the formation of senescence associated heterochromatin foci (SAHF), although it does not bind DNA domain. Nothing is known about its regulation and cellular localization in different cell types. I was wondering if someone is aware of a a good literature review on HP-1 and cellular senescence.

    Constantine Kaniklidis · No Surrender Breast Cancer Foundation (NSBCF)

    Manlio:

    There are a number of important papers1-19 in this fascinating arena - in addition to more general reviews12-21 - so I will briefly outline below some of the more relevant literature, and references within these will allow you to pursue this vital topic further.

    Cells from aged humans experience a decline in the levels of the heterochromatin 1 proteins (HP1) which are a component of senescence-associated heterochromatin foci (SAHF)1,2. In this connection, important are the results of the recent University of Rochester genetic study of heterochromatin formation in Drosophila3 that strongly demonstrate that heterochromatin levels - with HP1 being an integral component of heterochromatin and in fact controlling heterochromatin levels4,5, which in turn control rRNA synthesis [Larson, above] - significantly influence and prolong life span, with moderately higher levels of heterochromatin promoting longevity, given that it is confirmed that heterochromatin levels decrease with normal aging, and that heterochromatin formation is essential for silencing rRNA transcription (this transcription promoting protein synthesis in general) via repression of unnecessary rRNA synthesis in order to promote genome stability, and longevity.

    And investigators at the Fox Chase Cancer Center have demonstrated that HP1 proteins, prior to incorporation into SAHF, are transiently localized to PML bodies, where they colocalize with the chromatin regulator/histone chaperone HIRA6. More importantly, recruitment of HIRA into PML bodies is one of the earliest steps towards senescence in human cells [6], and we know that blocking entry of HIRA to promyelocytic (PML) bodies prevents formation of SAHF7, and HP-1 is centrally involved in the formation of ALT-associated PML nuclear bodies (APBs)8 playing a role in telomere-associated senescence.

    The Cancer Connection:
    Along a parallel line of inquiry, many investigators have also pursued the implications of HP1 and SAHF in the oncology context, leading to the hypothesis that the role of SAHF is to counter DNA-damage response (DDR) in order to force the cell into senescence (as opposed to apoptosis) and suggests that SAHF-associated heterochromatinization might be a barrier to tumorigenesis, as suggested by the fact that several cancer cell lines display elevated HP1 levels9. Therefore, opposing senescence-associated changes in heterochromatin HP1 (and H3K9) occur, consonant with the fact that age is a significant risk factor for cancer10.

    Narrowing the Role of SAHF/HP1:
    However, against all these views, it is critical to note that recent results11 have marshaled evidence that, unlike the DNA damage response marker γH2AX, SAHF is itself not a common feature of cellular senescence - indeed, that SAHF formation is wholly dispensable, and NOT, as commonly first thought, a universal component of senescence, for multiple forms of cellular senescence - and despite the fact that SAHF formation is shared by diverse cultured cell types under oncogenic stress, it nonetheless appears that SAHF are cell-type-restricted under genotoxin-induced and replicative senescence, and that in consequence, again against widely assumed views, that SAFH does NOT represent a reliable common marker to identify cells undergoing senescence in general (it would appear that only γH2AX-positivity, marking constitutive DNA damage signaling, is a reliable senescence marker), leaving a more narrow purpose for SAHF formation in oncogene-induced senescence.

    Where We Stand, and Going Forward
    Thus, we have evolved to a more nuanced understanding of the more limited role of SAHF and heterochromatin 1 proteins (HP1) in cellular senescence, although it nonetheless remains true that the heterochromatin process is vital, through elevated heterochromatin levels, in promoting genome stability, and longevity via silencing rRNA transcription and thus the repression of unnecessary rRNA synthesis, and it is clear that we will surely have other studies pursuing the exciting field of how heterochromatinization can be modulated to induced prolongation of lifespan, and - in parallel - suppression of tumorigenesis, thus advancing positivity both against aging and cancer.

    References

    1. Scaffidi P, Misteli T. Lamin A-dependent nuclear defects in human aging. Science. 2006;312:1059–63.
    2. Cao K, Capell BC, Erdos MR, Djabali K, Collins FS. A lamin A protein isoform overexpressed in Hutchinson-Gilford progeria syndrome interferes with mitosis in progeria and normal cells. Proc Natl Acad Sci USA. 2007;104:4949–4954.
    3. Larson K, Yan SJ, Tsurumi A, et al. Heterochromatin formation promotes longevity and represses ribosomal RNA synthesis. PLoS Genet 2012; 8(1):e1002473.
    4. Grewal SI, Elgin SC (2002) Heterochromatin: new possibilities for the inheritance of structure. Curr Opin Genet Dev 12: 178–187.
    5. Ebert A, Schotta G, Lein S, Kubicek S, Krauss V, et al. (2004) Su(var) genes regulate the balance between euchromatin and heterochromatin in Drosophila. Genes Dev 18: 2973–2983.
    6. Zhang R, Poustovoitov MV, Ye X, Santos HA, Chen W, et al. (2005) Formation of MacroH2A-containing senescence-associated heterochromatin foci and senescence driven by ASF1a and HIRA. Dev Cell 8: 19–30.
    7. Ye X, Zerlanko B, Zhang R, Somaiah N, Lipinski M, et al. (2007) Definition of pRB- and p53-dependent and independent steps in HIRA/ASF1a-mediated formation of senecence-associated heterochromatin foci. Mol Cell Biol 27: 2452–2465.
    8. Jiang WQ, Nguyen A, Cao Y, Chang AC, Reddel RR. HP1-mediated formation of alternative lengthening of telomeres-associated PML bodies requires HIRA but not ASF1a. PLoS One 2011; 6(2):e17036.
    9. Di Micco R, et al. Interplay between oncogene-induced DNA damage response and heterochromatin in senescence and cancer. Nat Cell Biol. 2011;13:292–U244.
    10. O'Sullivan RJ, Karlseder J. The great unravelling: chromatin as a modulator of the aging process. Trends Biochem Sci 2012; 37(11):466-76.
    11. Kosar M1, Bartkova J, Hubackova S, Hodny Z, Lukas J, Bartek J. Senescence-associated heterochromatin foci are dispensable for cellular senescence, occur in a cell type- and insult-dependent manner and follow expression of p16(ink4a). Cell Cycle. 2011 Feb 1;10(3):457-68. Epub 2011 Feb 1.
    12. Burgess RC, Misteli T, Oberdoerffer P. DNA damage, chromatin, and transcription: the trinity of aging. Curr Opin Cell Biol 2012; 24(6):724-30.
    13. Krishnan V, Liu B, Zhou Z. 'Relax and Repair' to restrain aging. Aging (Albany NY) 2011; 3(10):943-54.
    14. Lazarus A, Banerjee KK, Kolthur-Seetharam U. Small changes, big effects: chromatin goes aging. Subcell Biochem 2013; 61:151-76.
    15. Feser J, Tyler J. Chromatin structure as a mediator of aging. FEBS Lett 2011 Jul 7; 585(13):2041-8.
    16. O'Sullivan RJ, Karlseder J. The great unravelling: chromatin as a modulator of the aging process. Trends Biochem Sci 2012; 37(11):466-76.
    17. Chung I, Osterwald S, Deeg KI, Rippe K. PML body meets telomere: the beginning of an ALTernate ending? Nucleus 2012 May-Jun; 3(3):263-75.
    18. Corpet A, Stucki M. Chromatin maintenance and dynamics in senescence: a spotlight on SAHF formation and the epigenome of senescent cells. [Chromosoma 2014 May 27.
    19. Klement K, Goodarzi AA. DNA double strand break responses and chromatin alterations within the aging cell. Exp Cell Res 2014 Sep 8.
    20. Tsurumi A, Li WX. Global heterochromatin loss: a unifying theory of aging? Epigenetics 2012; 7(7):680-8.
    21. Campisi J. Aging, cellular senescence, and cancer. Annu Rev Physiol. 2013;75:685-705. doi: 10.1146/annurev-physiol-030212-183653. Epub 2012 Nov 8.
  • Reza Fazlalizadeh added an answer:
    What anesthetic can I use to bleed rats/mice from the orbital plexus?
    I am interested in doing retro orbital bleeding of mice/rats for doing FACS analysis of the whole blood. I am looking at a rare population. Right now I am using chloroform to do the same but I am not sure whether chloroform affects the cell numbers. Please tell me if chloroform influences the cell counts or any other parameter in the rodents and also suggest alternate and more reliable anesthetics.
    Reza Fazlalizadeh · University of California, Irvine

    You can find your answer here: http://web.jhu.edu/animalcare/procedures/retro-orbital.htmlA.

    Anesthesia

    Anesthesia is required for retro-orbital bleeding.The following anesthetic agents are recommended for this procedure:

    Mice - Ketamine (90 mg/kg) and Xylazine (10 mg/kg), IP
    Pentobarbital (50mg/kg), IP
    Proparacaine (Ophthetic®) - 1 drop per eye
    Isoflurane (Drop Method) - contact vet staff

    Rats - Ketamine (75 mg/kg) and Xylazine 10 mg/kg), IP
    Pentobarbital (50 mg/kg), IP

    Topical anesthetic, alone, may only be used in mice. Rats, who require more restraint due to their size, must be bled under general anesthesia. Note that there is the potential for contamination of the blood sample if topical anesthetic is used.
    Use of any other anesthetic agents must be identified in the IACUC application.

  • Carlo Palesi added an answer:
    Does chlorhexadine affect the viability of thawed stem cells?

    Exploring a new catheter which is coated with chlorhexidine for stem cells infusion.

    Carlo Palesi · Montefiore Medical Center

    Thank you!!

  • Michael ER Olausson added an answer:
    What is the difference between cell engraftment and repopulation?

    What is the difference between cell engraftment and repopulation?

    Michael ER Olausson · University of Gothenburg

    I do not see the difference. If you have a malfunctioning tissue it can either be caused by cells missing due to specific targeted destruction, or a by a general destruction. If you believe that remaining signaling proteins in the ECM could "home" the cells needed, there is really no difference. The "homing" signal comes from the matrix proteins, either called repopulation or engraftment. If we want to differentiate between these entities, I would define engraftment the process of producing more than the original blueprint.

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