Stem Cell Biology

Stem Cell Biology

  • Sihan Wu added an answer:
    How can I improve thawing efficiency of human iPSC?

    Hi everybody. I've bought human CD34+ derived iPSC line from Life Technologies. In the initial recovery as soon as I got the cells, it worked great. However when I cryopreserved the cells with E8 medium with 10% DMSO and thaw them, I found massive cell death.

    Here are the details:

    Day 1. Thaw cells as fast as possible. Culture them with E8 medium + ROCK inhibitor.

    Day 2. Most of the cells are viable. Change the medium, with ROCK inhibitor.

    Day 3. Start to see modest cell death. There're still some decent looking colonies. Change the medium, with ROCK inhibitor.

    Day 4. Massive cell death. Although there're still some cells left (but not quite look like colonies) adherent in the dish (vitronectin coated), I am worry that eventually all the cell will die.

    So is that normal or I did something wrong with the culture? I will definitely try longer culture.

    Sihan Wu · Ludwig Institute for Cancer Research

    Thank Yanuar and Prasad

  • Siamak Gholamalipour added an answer:
    Is there anyone with experience in using and successfully differentiating the Rat Fetal Neural Stem Cells from Invitrogen into Oligodendrocytes?

    I managed to get the cells to reach confluence, and I fixed and applied primary Antibodies of CNPase and MBP about 8-9 days after the initial addition of the Differentiation Media, however when I imaged them, there as no positive staining for either CNPase or MBP. I was wondering if there are any tips or tricks about how long to wait before going in to characterize, or any antibodies that have worked well. 

    I was also interested in whether anyone has been able to succesfully re-thaw P1 generation cells that were frozen down in DMSO and kep in liquid nitrogen. I seem to be having problems with the cells clumping together after thawing them again, and also very low viability. Any tips or tricks for this issue?

    Siamak Gholamalipour · Urmia University

    Dear Erika please check this article

    i hope it's helpful.

  • Lucie Farrera added an answer:
    How can I dissolve EGTA
    I need to prepare 5mM but it's too difficult to dissolve the EGTA
    In PBS. I'm Use it for some types of stem cells just for dissociate the cells
    Lucie Farrera · Université Montpellier 2 Sciences et Techniques

    This topic helped me very well , thank you !!

  • Kenneth Ka-Ho Lee added an answer:
    Has anyone tried to repeat the Hou et al.,2013 Science 341:651-651 experiment of using only seven small molecules to produce ciPS cells?

    It is now 2 years since the publication of this very important study by Hongkui Deng.  Has anyone managed to reproduce it?

    "Pluripotent Stem Cells Induced from Mouse Somatic Cells by Small-Molecule Compounds"
    Pingping Hou, Yanqin Li, Xu Zhang, Chun Liu, Jingyang Guan, Honggang Li, Ting Zhao, Junqing Ye, Weifeng Yang, Kang Liu, Jian Ge, Jun Xu, Qiang Zhang, Yang Zhao, Hongkui Deng, Science (2013)

    Kenneth Ka-Ho Lee · The Chinese University of Hong Kong

    Hi Tomo,

    Maybe you should consider publishing you negative results.  Journals like F1000Research will publish it - probably not to the liking of Science.

    Ken

  • Monica Yiyun Jiang added an answer:
    Can I put 2 promoters together and hope both of them will function?

    Hi All,

    As my problem, I am working with ES cell lines and want to induce them into neurons. in this process we want to observe it as GFP+ cells. but seems the promoter (i.e ef1a) express high in es cell didn't working well in neurons, and neuron specific promoter didn't working in ES cells. 

    So I was thinking make a plasmid, has 2 promoter. like this:

    neuron promoter-es cell promoter-GFP-polyA.

    So that in es cell stage, the es cell promoter can working, and in neurons, the neuron specific promoter can working. but I didn't see any plasmid like this. 

    Does anyone do this before?

    Many thanks

    Yiyun

    Monica Yiyun Jiang · Wayne State University

    Ok seems all of reply said need two promoter with two protein, and Paritosh's idea is great, so that I can see the process from ES cell to neurons. Thanks all of yours!

  • Indumati Sharma 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.

    Indumati Sharma · University of Auckland

     How effective is RetroNectin?

  • Clara Grudina added an answer:
    What is a suitable method to freeze hIPSC on feeders and on matrigel?

    I don't know if there are some differences in freezing between IPSc on feeders and IPSc on matrigel. What type of recipe do you use? And during the defreezing what is the percentage of dead cells? Thanks in advance.

    Clara Grudina · Scuola Internazionale Superiore di Studi Avanzati di Trieste

    Thank so much!! Unfortunately I left some days at -80°C I hope not to compromise everything...

  • Sravan Goparaju added an answer:
    Can anyone help with neuron attachment on glass coverslips?
    Been differentiating ES/IPS cells into neurons. I need to culture them on glass coverslips for downstream microscopy. I find that neurons don't attach well to coverslips coated with Poly-Lysine, PL/Laminin and PL/LM/Fibronectin combinations. LM alone, coating looks OK except that the attachment is not strong! So, whenever I change medium invariably some cells come off the surface and by the end of incubation period over a couple of weeks many of the cells are gone. Any ideas to get a more firm attachment of neurons to glass coverslips?
    Sravan Goparaju · Keio University

    All - Thanks a bunch for the suggestions. I could optimize the conditions for the neurons I work with (Motor neurons). Coating with Ornithine for 2 hrs followed by overnight coating with Laminin at a relatively high conc of 20 ug/ml allowed MN to attach well and remain stable for at least a week. Sometimes I find some neurites - kind of floating around at which time I add additional laminin 1-2 ug/ml made up in cell culture medium which seem to help the neurites re-attach (read that in a published paper elsewhere) .

    @Monica Yiyun Jiang - Coating with gelatin did not help. Cells did not attach. 

  • Yon Jin Chuah added an answer:
    Can anyone tell me if mesenchymal stem cells can attach on plain PLGA scaffolds without any coating?

    I am planning to do some study on mesenchymal stem cells seeded on PLGA based-scaffold. I found in the literature both manuals that are using collagen coating and that are not specifying any coating process. It would be very helpful to know if any coating is needed or not in order to design the scaffold properly.

    Thank you in advance

    Yon Jin Chuah · Nanyang Technological University

    As mentioned by many, native PLGA may not perform too well as compare to those which are treated. Recently, I have published some surface treatment methodology to improve cell adhesion, proliferation and even differentiation on certain polymer. You may refer to that for more information.

    http://pubs.rsc.org/en/content/articlelanding/2015/bm/c4bm00268g#!divAbstract

  • Thomas Andl added an answer:
    Why does cell cycle quiescence lead to maintenance of stem cell capacity?

    Thank you.

    Thomas Andl · Vanderbilt University

    Interesting question. Why do you think there is a causative relationship between quiescence and maintenance of stemness? In vitro, the quintessential stem cells, embryonic stem cells, are highly proliferative. In vivo, this may be different and there is good reason to assume that quiescence protects stem cells from accumulating critical levels of DNA damage. But is this for real? In my opinion, stem cell quiescence is only relevant for organisms with a long lifespan AND tissues with high turnover (most epithelia, bone marrow). In short-lived animals there seems to be less pressure to keep the stem cells quiescent. And if you check out rodent label retaining cell experiments, there is not much label retaining. But there is not much hard data to show that high proliferation in the stem cell compartment is bad, especially for long-lived animals.

    In the colon, even in mice, they favor now the two state model of stemness: the quiescent and the active stem cells (Li et al. Single-cell analysis of proxy reporter allele-marked epithelial cells establishes intestinal stem cell hierarchy. Stem Cell Reports. 2014 Nov 11;3(5):876-91.)

    So, your question is intriguing and there is some evidence that a slow cycling stem cells are good on the long run, but I am not sure that there is more to it than common sense believe. If stem cell quiescence is a key to maintain a tissue over long periods of time without increased cancer risk then epithelia of long-lived animals with high cellular turnover should look very different from epithelia of short-lived animals. Isn’t that the logical conclusion? And if not, how would long-lived animals address the problem of stem cell proliferation over decades versus short-lived animals over months or a few years (e.g. humans, whales, elephants versus rodents)? Isn’t this what is summarized with the term Peto’s paradox (http://en.wikipedia.org/wiki/Peto's_paradox; onlinelibrary.wiley.com/doi/10.1111/eva.12228/abstract; http://www.cell.com/trends/ecology-evolution/abstract/S0169-5347(11)00015-2?_returnURL=http%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS0169534711000152%3Fshowall%3Dtrue)?

    The important concept here could be that reduction of cell proliferation is a key to prevent cancer. This is a nice hypothesis and I would be very interested to see hard evidence that change in stem cell proliferation rates correlates with longevity and cancer risk. In the mouse, we may see with the “two-stem-cell model” in a highly proliferative tissue the dawn of this new concept. I would not called it “two-stem-cell” since in vivo under homeostatic conditions there might be a strict hierarchy that makes the quiescent stem cell the top dog and the other one the work horse (isn’t that one technically a transient amplifying cell?).

  • Prasad Pethe added an answer:
    Would you rather use knockout or heat inactivated serum to expand MSCs?

    I am trying to decide what serum to use to expand canine mesenchymal stem cells and/or mesenchymal stem cell progenitor cells without losing stemness aka. avoiding spontaneous differentiation. Thanks.

    Prasad Pethe · National Institute for Research in Reproductive Health (NIRRH)

    Using Knock out serum replacement with recombinant proteins would be ideal, but expensive. You can use heat inactivated FBS to expand MSCs, but keep regularly checking the protein and RNA profile to ensure that MSCs are not differentiating. On Knockout serum, it tends to more BMP, which can disrupt some cells types. Good luck with using heat inactivated FBS.

  • Anne Leferink added an answer:
    Do mesenchymal stem cells form embryoid bodies?

    I am trying to isolate umbilical warthon's jelly MSCs and I find some populations that forms embryoid body like clusters. I have never seen this ones before when working with AD-MSC. I am wondering if it is a different population.Thanks

    Anne Leferink · Maastricht University

    You could check the work of Erik Vrij and Nicolas Rivron (group op van Blitterswijk). They are working on embroyonic bodies/cavitation/blastocyst fromation etc. Maybe that helps?

  • Bianca Völkening added an answer:
    How long and which concentration of KCl do I need to depolarize NPCs if I want to measure afterwards the proliferation?

    I have Neural stem cells (derived from adult mouse HC) in a neurosphere culture. As I saw changes in proliferation in my knockout in vivo after depolarisation, I would like to repeat this experiment in vitro. 

    I would like to depolarize the cells with KCl and afterwards do a BrdU-proliferation-assay. I want to see the changes in the type1 cells - not in differentiated neurons. I would first plate the dissociated neurosheres on cover slides coated with matrigel (in medium) over night to let them settle down and then change the medium with medium containing KCl.

    But I find very different protocols concerning concentration of KCl (between 15 and 100um) and duration (6h to 6days). 

    Do you have any suggestions on wich concentration and duration I should use?

    Thanks!

    Bianca Völkening · Central Institute of Mental Health

    Thank al lot Kathleen! As I don't have so many cells I have to check if I can try all at once, but that is a good suggestion :) 

  • Hazem Ghebeh added an answer:
    How do you analyse FACS data obtained by the ALDEFLUOR kit?
    How do you analyse the FACS data obtained by the ALDEFLUOR kit? Where do you set the gate on the inhibited sample (DEAB)? What should be the cut-off on the inhibited sample? If you set the ALDHhi gate really close to the cell popluation in the DEAB sample then you have a rather high intra-experimental variation.
    Look forward to hear from anybody who worked with this kit.
    Hazem Ghebeh · King Faisal Specialist Hospital and Research Centre

    You need both methods to analyze your results, especially if you are measuring the effect of a drug for example, and yes the range might be very small but FACS by its nature is very sensitive and is an accurate method so if results are reproducible with different replicates then it is true.. Whether that little shift reflect a change in stem cell activity or not, this is a different question that can be answered in combination with other experiments.

    Also you should be careful with treatment using drugs that have intrinsic natural fluorescence as they might interfere with the assay unless you compensate for that.

    By the way, while using ALDEFLUOR kit, FACS measures total ALDH activity and not just ALDH1 as probably all the isoforms of this enzyme can do the reaction and not just ALDH1 (this is an enzymatic method and not an immunological assay).

  • Alessia Vivanti added an answer:
    What is the proportion of cells in human bone marrow?

    I'm looking for percents of several kind of cells in bone marrow and the range of proportion per mL from an aspirate.

    I would like to see the differences in first stages of leukemia, lately stages of leukemia and healthy people (it seems more difficult, but maybe before the transplant this info is available). 

    It's related with my Master thesis project, and I'm centering not only in stem cells, also in progenitor cells. 

    Alessia Vivanti · Stem Cell Medicine Ltd

    These two papers may be useful: 

    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3269474/pdf/0970179.pdfhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3269474/pdf/0970179.pdf

    http://cdn.intechopen.com/pdfs-wm/42803.pdf

  • Christian Unger added an answer:
    Is there anyone with expert advice on IPSC derivation from MEFs using episomal vectors?

    I've been recently trying to derive iPSCs from low-passage MEFs using episomal vector electroporation (3 vectors: hOCT4-p53shRNA; hSOX2/hKLF4; and hN-MYC/hLIN28 - (h=human)).

    Because I need to analyze the effect of a certain agent on reprogramming efficiency, I prefer to use the 2-vector combination (OSK) to induce iPSC generation, because OSKML appears to mask the enhancing effect of other 'minor' reprogramming agents.

    The problem is that when I evaluate two combinations (OSK and OSKML) for iPS generation, cells electroprated with OSK undergo massive cell death, while those with OSKML do not display a high death rate and grow normally. So I couldn't derive iPS from OSK combination, and could derive one single iPSC colony per 12000 cells initially seeded with OSKML cocktail.

    Does anyone of you have any expert advice on my work? I need details. Any other related comments would also be appreciated.

    Thanks in advance,
    Sharif

    Christian Unger · The University of Sheffield

    Just a few additional points to the previous comments. It is an expected results that 3F without Myc will give you a significantly lower reprogramming efficiency. So your results are in line with that.

    To perhaps help you increase efficiency and counteract your toxicity I suggest you a simple addition of inactivated MEFs when you notice the toxicity. The toxicity occurs through high expression of the reprogramming factors and that can be beneficial to your reprogramming efficiency. However, when the expression really kicks in, the cells are in a very fragile state and that is when extra support cells can help. Just add them into your medium. Don't add too many though, just enough to keep a close to confluent cell layer. Too high density will also reduce your efficiency.

  • Penelope M Tsimbouri added an answer:
    Has anybody isolated protein from mouse mesenchymal stem cells? I am facing a problem with poor concentration of protein when using RIPA Lysis buffer?

    Shall I change to any other harsh lysis buffer? Also I have run the gel on SDS PAGE  I am getting one single band between 50- 75 Kda. Is it because of improper denaturation? Please help.

    Penelope M Tsimbouri · University of Glasgow

    hi,

    make sure you work on ice for all steps. Make sure your buffer if not very old and inhibitors are added just prior to use.

    I tend wash my cells first a couple of times with cold 1xPBS to get rid off any medium proteins.

    I tend to trypsinise my cells and pellet them in an eppendorf tube prior lysis to ensure i can have my samples as concentrated as possible.

    RIPA should extract the majority of proteins. The fact you are using a cocktail of protease inhibitors does help.I tend to sonicate the samples to get rib off any nucleic acid and spin down to pellet the debri. Then transfer the supernatant into a new tube.

    After measuring the protein concentration I aliquot the required amount into a tube and add protein loading buffer. 

    depending on what you are using it for 20-30µg/lane is good for an SDS page. However, it may also depend on the abundance of the protein you are after.

    I denature my samples in reducing protein buffer containing beta-mercaptoethanol and denature the proteins by heating at 98C for 2min. then put back on ice to cool down, spin down briefly to collect sample prior loading.

    I hope that helps

    good luck

  • Syed Raza added an answer:
    Do anyone have experience with single cell cloning of neural stem cell/progenitor cell?

    I have to select my CRISPR KO cells which will show the best result from NPCs. For the same I wish to do single cell cloning for NPCs, but I am afraid that single cell cloning may lead to differentiation of NPCs? 

    Does anyone have experience of single cell cloning with NSCs/NPCs? 

    Please advice.

    Thanks in Advance,

    Syed

    Syed Raza · Cleveland Clinic

    Thanks Daria

  • Véry Coulic added an answer:
    Is it possible to "deconstruct " an adult stem cell into its constituent sperm and egg cells?

    I have been wondering for quite some time about this: if an egg cell and a sperm cell are used to create a zygote that then divides and gives rise to many stem cells, what is preventing the zygote from being deconstructed so that we can form both an egg cell and a sperm cell from it and is this therefore even theoretically possible? Considering the fact that the egg cell has DNA that codes for the egg, in addition to other epigenetic factors, and so too does the sperm cell code for the shape and design of the sperm cell, and this information is then present in the fertilized cell, why wouldn't we be able to isolate the DNA or reform the constituent egg and sperm cells from stem cells (for instance, if we label the maternal and paternal chromosomes in each gamete then we separate them after fertilization and then attempt this deconstruction)? Thank you!

    Véry Coulic · Université Libre de Bruxelles

    The question is interesting, bit is it correctly formulated? What do want exactly: to restore theovuleand the spermatozoid from a zygote, or to obtain separate ovule DNA and spermatozoid DNA? Because ovule is a formation indeed quite different from spermatozoid but both are also qualitatively differe from the egg. Thezygote has lost a part of the spermatozoid. Do you want to recover it? how? and so on.

    Good luck

  • Sofiane Hamidi added an answer:
    How do I keep my mesenchymal stem cells isolated from human umbilical cord in an undifferentiated state?

    Currently I am working on isolation of mesenchymal stem cells from human umbilical cord. Since MSCs have the unique properties of self renewal and differentiation into various lineages, my protocol demands to keep them undifferentiated. Nanog and LIF have been mentioned as factors responsible for undifferentitation of MSCs. However their roles are not clear. Kindly help.

    Sofiane Hamidi · French Institute of Health and Medical Research

    Hi

    I totally agree with previous comments, be careful to passage them before they reach critical confluence or they will differentiate/ undergo apoptosis.

    That's one of the main critical point.

    Good luck

  • Duccio Lombardi added an answer:
    How can I trace transplanted Stem Cells in vivo, which are not labelled for any color?

    Hi All, 

    A friend of mine from India has raised a concern since they don't have the transduction facility, what are the other ways to trace the transplanted Stem Cells in vivo in rodents if they are not virally transduce for any color e.g. GFP/mCherry etc. Their lab is planing to transplant the mouse NSCs in mouse only. 

    Kindly please suggest.

    Many thanks,

    Syed

  • Henry E Young 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.
    Henry E Young · Mercer University

    Dear Dr. Tamaki,

    We used multiple markers (109) to identify 66 distinct cell types, based on phenotypic expression. The particular markers used are listed in Table 1 of the MOJ 2014 article.

  • Ceren Günes 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?

    Ceren Günes · Technische Universität Dresden

    Thanks for alll the answers. I used Matrigel and the reprogrammed iPSC grow just fine!

    Glen, if you also reprogram CD34+ HSC, there is a new reprogramming medium from Stem Cell Technologies, called ReproTeSR. I tried it in parallel to StemPro, and some iPSC like colonies emerged as of 1st week already, I guess I'll stick to it for future experiments!

    http://www.stemcell.com/en/Products/All-Products/ReproTeSR.aspx

  • Henry E Young added an answer:
    Adult Stem Cell differentiation: is stemness the default pathway?
    On in vitro differentiation assays using adult stem cell cultures, do the adult stem cells differentiate, or are only the early and late progenitors the only ones that differentiate? in other words is stemness the default pathway? does somebody know any examples where it has been proven that the stem stay?
    Henry E Young · Mercer University

    I would disagree with the comment about MSCs. But then again, it depends on which MSC you are discussing, i.e., marrow stromal cells, mesenchymal stem cells, or mesodermal stem cells. We started with a population of mesodermal stem cells cloned from a single cell by repetitive serial dilution clonogenic analysis. So far we have expanded the clone through 690 population doublings without them undergoing either spontaneous differentiation or mutation of their karyotype. If we want cells from that clonal population to differentiate, we remove aliquots of stem cells and induce them with various boioactive agents for mesodermal cell types, such as BMP-2, VEGF, TGF-beta, Basic-FGF, etc. One can also use dexamethasone at concentrations from 10-6 to 10-10 M and demonstrate multiple mesodermal lineage cell types. We have been able to differentiate this clone of mesodermal stem cells into muscle, fat, cartilage, bone, tendon, ligament, dermis, scar tissue, capsule, trabeculae, endothelial cells, blood cells, etc.

  • Christophe Lancrin added an answer:
    If the STAP phenomenon is found to be real, what will be the impact on biology?
    Right now, the jury is still out on STAP cells. It is not known for sure if STAP (stimulus-triggered acquisition of pluripotency) is a genuine phenomenon. Top laboratories around the world, in Japan, the United States and elsewhere, are working to try to replicate the findings of Obokata et al. (Nature 505:641-647; Nature 505:676-680). As they do this, I sincerely hope they take the sage advice of Hans Scholer, who suggested that RIKEN (the main team who is attempting to replicate the work) involve Obokata in each and every step of the procedure (see Nature 508:299). Obokata insists that STAP is real. This may be the case, and it may be that the procedure is very tricky; she might have succeeded where others have failed because she has apparently spent every waking moment for years working out the details. This is our (the scientific community's) chance to really get to the bottom of this issue. If we blow this opportunity, we may never know the answer to this important question. If we're not careful, this moment could pass us by.

    The reason why it is so important to know if STAP is real is because, if it is, then this will be a paradigm-shifting discovery. In the 1990s (and even earlier, through the work of Sir John Gurdon) we learned that the cytoplasm of an enucleated oocyte can reprogram a nucleus from a terminally-differentiated somatic cell that has been transferred into it (using somatic cell nuclear transfer, SCNT) to a plurpotent state. More recently, in 2006, we learned that a limited set of four factors, the so-called Yamanaka factors, can also reprogram somatic cells to pluripotency, producing iPS cells. These were astounding discoveries, ones that told us something remarkable about the nature of the cell: that it has the capacity to "reverse course" and become a pluropotent stem cell under certain conditions. If STAP is real, then this means that the conditions under which this sort of thing can occur are even broader than we ever could have imagined, to also include external triggers (low pH, mechanical stress). What would this tell us about nuclear-cytoplasmic interaction and the role of the whole cell in gene expression patterns? How would the human body (for instance) normally prevent this kind of reversion to pluripotency, which would potentially lead to cancer? These are exciting questions. Let's get this thing right; let's pursue all avenues to find out what the real answer is!
    Christophe Lancrin · European Molecular Biology Laboratory

    I am very skeptical about this STAP cells. Read this excellent article about the case. This is very informative: http://www.theguardian.com/science/2015/feb/18/haruko-obokata-stap-cells-controversy-scientists-lie

  • Wassim Eid added an answer:
    Has anyone used SNL feeders for rat iPSC derivation?

    Used as source of Lif?

    Great discussion board! 

    Wassim Eid · Université de Fribourg

    I know it is an old question, you may have found an answer by know, but I add this information for any one interested in the future. Yes it seems you could (according to the study attached) I am not sure though if they are enough to be used as source of Lif.

  • Robyn Meech added an answer:
    Can anyone help with mammosphere quantification?
    I'm having trouble quantifying the weekly results of my mammosphere protocol for measuring stemness in cancer cell lines. Using StemCell's protocol, on the day of counting I:
    harvest the mammosphere + media
    Spin down
    Resuspend in 500uL of mmaosphere media
    Transfer 50uL to a 96 well and count the numbers in this dilution

    The main problem is that when I get to the 96 well, I've been seeing quite a reduction in the size of my mammospheres. I'm worried that the slight manipulation of the spheres leading up to this are breaking them up. But its also impossible to count if I were to keep the spheres in their normal 6-wells.

    Does anyone have any experience or know people who have somewhat mastered mammospheres and could help me figure out how to better quantify? I could really use some help.
    Robyn Meech · Flinders University

    Have you tried coating with 1.2% polyhema instead of agar? It works well for us in larger wells (24 , 48 well) but I haven't tried 96 well.

  • 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.

    Christopher Daniel Duntsch · Hybrid Bioscience, Inc., Synthetic Investments, Inc., The University of Tennessee Health Science Center

    Below is the citation and abstract for a relevant article to the question. And while I like this manuscript, it is interesting how it relates in different ways to the above answers. All of which are well done in their own right. The article is attached.

    Stem Cells

    2010;28:75–83 - www.StemCells.com

    EMBRYONIC STEM CELLS/INDUCED PLURIPOTENT STEM CELLS

    Characterization of Gastrulation-Stage Progenitor Cells and Their Inhibitory Crosstalk in Human Embryoid Bodies

    ODED KOPPER, ODIL GILADI, TAMAR GOLAN-LEV, NISSIM BENVENISTY

    Stem Cell Unit, Department of Genetics, The Institute of Life Sciences, The Hebrew University, Edmund J. Safra Campus, Givat Ram, Jerusalem, Israel

    Key Words. Embryogenesis • Human embryoid bodies • Human embryonic stem cells • Progenitor cells

    ABSTRACT Human embryoid bodies (HEBs) are cell aggregates that are produced during the course of embryonic stem cell differentiation in suspension. Mature HEBs have been shown to contain derivatives of the three embryonic germ layers. In this study, using a combination of laser capture microscopy followed by DNA microarray analysis and cell sorting, we demonstrate that early HEBs are composed of three major cell populations. These cell populations can be defined by the expression of specific cell markers, namely: (i) OCT41, REX12; (ii) NCAD1, OCT42; and (iii) EPOR1, OCT42. By analyzing gene expression in embryonic tissues, these cell populations could respectively be assigned to the embryonic ectoderm, mesendoderm, and extraembryonic endoderm lineages. We show that the extraembryonic endoderm, which selectively expresses platelet-derived growth factor B (PDGF-B), negatively affects the mesendoderm lineage, which selectively expresses the receptor PDGFRA. Our analysis suggests that early HEBs are spatially patterned and that cell differentiation is governed by interactions between the different cell types.

  • Mitul Vakani added an answer:
    Why do I get settled down Islets during Islet differentiation from human Bone marrow mesenchymal stem cells ?

    During Islets differentiation process, most of Islets get settled down into non adherence culture plates. So that i didnt get mature Islets because its staring to  de-differentiation. Ideally i want floated  Islets through out Islet differentiation Process.

    Mitul Vakani · The Maharaja Sayajirao University of Baroda

    my culture media condition for differentiation  DMEM KO + 1 % BSA + Activin A ( as differentiating agents) [5ng/ml]

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