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Stem Cell Differentiation - Science topic

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Hello! I currently have differentiated iPSCs I wish to clean up. I use mTESR with matrigel coating.
ReLeSR selectively dissociates healthy stem cells, however even on quick release (1 minute), all the cells immediately dissociate, and I am unable to isolate healthy stem cells.
I think that it might be due to me passaging very regularly (once every three days), and the cells have been adapted and now dissociate easily.
I would also like to attribute that to the reason why my iPSCs are differentiated. I supplement them with ROCK inhibitor (5uM) every time I passage. Given that they are passaged once every three days, that means they are in ROCK inhibitor once every two days... could this be the reason my iPSCs are so differentiated, as I did not give them sufficient time to recover from ROCK inhibitor?
However, the reason why I passage so regularly is because I know need to passage once I see differentiated cells... Do you suggest I completely omit ROCK inhibitor during passaging, and would it help the maintenance of my iPSCs? For individuals with experience using ROCK inhibitor during passaging, do you feel that frequent passaging causes spontaneous differentiation of iPSCs even when you remove ROCK inhibitor the next day (before 24 hours)?
I would really appreciate any advice that would help me out of this predicament, thank you so much!
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Hi,
The incubation time with ReLeSR is too long. You need to optimize your incubation time. Seed iPS colonies at a much lower density.
If the iPSC colony confluence is very high on the passage day, ReLeSR reagents may not be able to distinguish between differentiated and undifferentiated cells. You need to increase the time between passages and reduce the iPSC seeding density.
Part of the iPSC lineage is very unstable in terms of phenotype. This means that cleaning must be done regularly. Sometimes by regular manual removal of colonies with incorrect morphology (every day). Additionally, they use ReLeSR before the passage should be performed.
If a high number of differentiated iPSC colonies is observed, positive selection should be used.
At the beginning:
- use a low seeding density (e.g. 20 most beautiful colonies (positive selection) per well of a 6-well plate);
- extend the time between passages before applying ReleSR (iPSC colonies should be large, highly compacted, in confluency 60-70%);
- optimize the incubation time with ReLeSR;
- use DPBS without calcium and magnesium;
ROCK inhibitor does not have to be used for every passage. It is recommended in the situation of iPSC thawing and positive selection only.
Good luck!
Justyna Augustyniak
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I am culturing human induced pluripotent stem cells for differentiation into cardiomyocytes via the Wnti-directed protocol attached here. After passaging into a 12 well plate to start the differentiation, the iPSCs should be 100% confluent the next day so that differentiation can be started. But it takes mine 2-3 days to reach full confluency, and they rapidly lose confluency as I start the differentiation protocol. By around day 5 of the protocol, they have essentially all died off. Unsure what I'm doing wrong and would welcome any theories or advice. I was using ECM Gel but am now using Matrigel. I use mTesR media with ROCKi added for the first 24h only.
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what is the concentration of Wnt activator and Wnt inhibitor you are applying to the cells? Maybe there is something that is going wrong here. If this is fine then my next suggestion would be to optimizing the seeding density and confluency at which you will start the differentiation for the particular cell line that you are using.
It is not necessary that the iPSC differentiation will work best/better at 100% confluency. In my experience, the best cell density for differentiation can vary amongst different iPSC lines.
Good luck with troubleshooting!
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Hi everyone,
So these are images of IPSCs 2 days post passaging/splitting. I noticed that the overall morphology of colonies are not compact or round and without distinct border. However, at higher magnification, i can still see cellular morphology of typical IPS cell with scnat cytoplasm and prominent nucleoli
Any help is much appreciated!
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Hello - (caveat - I am more familiar with ESC cells, rather than iPSCs; and your culture method choice will influence morphology subtly too), but these do look like they are differentiating (or at least stressed). The main comparison I'm looking at is the edge cells compared to the middle cells in the same aggregate - these look much more elongated and less immature than the center cells which is indicative of differentiation. However, you mention these are 2d-post-split, so it could just be stressed cells from splitting. The key here is if they round back out and make more compact&dense aggregates by ~4d post split (with likely some small areas of differentiation). If they do, then I would recomend adjusting your split technique to be a bit gentler to avoid this issue in the future - as stressed cells are much more likely to spontaneously differentiate (and depending on your downstream experiments, also less likely to behave properly there).
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My cells die a lot between day 4 and 5 of differentiation so I can't get a good number to proceed in the differentiation steps to hepatoblasts
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A short update: I had the problem that my cells were dying shortly after DE stage. I figured out two main reasons. 1. The seeding density is really crucial (as mentioned in every differentiation protocol) - Too many cells and your cells will form a layer that will detach - Too many cells means a drop of pH, which might cause cell death or destruction of your surface coating (e.g. Matrigel)
2. The addition of Penicillin / Streptomycin - As soon as I add Pen/Strep at the beginning of my differentiation, cells die. I also heard that from other people. Since then, I do the differentiation without Pen/Strep. I was told its okay to add the Pen/Strep to a later timepoint of the differentiation. So far, I never tried that.
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Dear all, anyone knows how to obtain pax7+/myoD- skeletal muscles progenitor cells (satellite cells) from mesenchymal stem cells? i am looking for and induction protocol without using transcription factors over-expression.
thank you ;)
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Have you tried Wnt3a treatment? it is critical in induction and maintenance of pax7, while inhibiting myoD, myogenin in segmental mesoderm.
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I am differentiating Rat fetal Stem cells into Neurons. I extract protein for Western blot and quantify them by BCA kit. The problem I am having is after incubation the color of my sample looks similar to a particular standard but when I read the plate using a plate reader the value I get is lower than the zero standard. Someone please help me with this.
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Mariana Marques, it depends on lots of things. First make sure your standards are in a good condition. If you are not sure about their conditions prepare freshly. Next thing is the plate you are using to measure, make sure you are using a new plate don't reuse plates, that might sometime interfere with the readings. And also make sure the recipe of your RIPA buffer (I assume you use RIPA buffer). The conc of DMSO more than 10% will also interfere with your results. Hope this helps you.
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Endometrium-derived mesenchymal stem cells changed morphology after IGFBP3 gene knockout via CRISPR/Cas9 plasmid. Selection of knockout cells was performed with puromycin. Apparently, unexpected differentiation has happened: knockout cells lost MSC markers (CD73, CD90, CD105, CD146), immunofluorescence microscopy also revealed that there is no more vimentin in knockout cells, so we suppose that is not osteogenic or chondrogenic/adipogenic differentiation. Nestin is absent in knockout cells too, consequently they are not neural progenitors. Cells appear to be terminal differentiated because of growth rate decreasing. Here are the questions:
1) What type of cell could it be? Do the cells look like epithelial culture?
2)What are these foci-like structures inside knockout cell nuclei? They are clearly distinguishable when stained with DAPI. 
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I sometime saw fibroblast like that after stressfull conditions, mainly tripsin excess. They keep growing but slower and eventualy stop and died after 2 or 3 passages.
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I want to develop a lab-made media for differentiation of ADSCs into Keratinocyte-like cells.
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Hello
I am planning to use human iPSC derived neural stem cells to differentiate and study Alzheimer's disease. For the immunofluorescence assay can I use Tyrosine Hydroxylase? or is it suitable only for differentiated dopaminergic neurons?
If so can I get a suggestion for any other marker?
Thank you in advance!
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David J Rademacher Thank you very much for the answer.
I simultaneously study Parkinson's Disease which uses TH in fluorescence imaging. However, I can use separate one for Alzheimer's.
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I'd like to make some Wnt/R-Sponding media to lower the costs of organoid maintenance
Many thanks
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I am learning to culture organoids. Many thanks for the updating information.
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I often wondering about nature !!!!
I seek nature to provide an opportunity to understand its beauty of its function.....after attending a great talk by few scientists physics and chemistry also plays a major role in biological function....
A great secret behind the function of cells in human starts since the sperm reaches the egg....
Can any one help me to understand my few ques as follows:
In a Zygote
1. what is the driving force of the embryonic stem cell to be differentiated into different kind of cells to form an organ?
2. Though all the cells have the same DNA, how nature decides what kind of cells are required for the function of an organ? and also how does cells know to function specific to the organ??
3.What make these specific kind of cells to function or express gene for specific organ???
3. Differentiated cells make each and every organ perfectly in a perfect shape... how does the cells know this is going to be shape of an organ and once the shape is achieved the cells proliferation is stopped, How?
All these things are happening within the maximum time period of 9 months... how mystery this nature is :)
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In simple words its the heterochromatin patterns which reflects cellular identity.
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I am differentiating Rat fetal Neural stem cells into Neurons. I used Nestin for stem cells in ICC and Western and I got the result I expected, but to confirm my differentiated cells as neurons I want use Tuj1. My doubt is whether Tuj1 express in both stem cells and differentiated cells and also I want to know more details on various Neuronal markers. Can anybody suggest me a solution?
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Tuj1 expressed only in diffrentiated cells. For stem cells yopu can use SSEA1 marker
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I am growing 2-D cell cultures for about 15-20 days as part of a stem cell differentiation protocol, and all of a sudden my cultures stopped dissociating into single cells when I use TrypLE or Acutase. I opened a bottle of new TrypLE and it still didn't work so it's not an issue of a faulty enzyme. The cells come off the plate just fine - they come off in one large sheet but will not dissociate into single cells. Anyone know why this is or how I can fix it? They would dissociate just fine before but now they don't.
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If your 2D culture comes off as a single sheet, let it be. Collect that sheet into a 15ml falcon tube, add about 2 ml of Tryple E express, or else 0.25% Trypsin, titurate using a pipet tip and allow to stand at 37 deg C for 3-5 minutes. You will be able to dissociate the sheets into a single cell suspension.
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Hi all,
I want to compare the gene expression of 5 genes in two groups (control group> undifferentiated stem cells and my test group > differentiated stem cells), I use graph pad to analyse. My question; what is the right statistical test to choose, if its Paired T-test how do I compute the data set in graph pad.
As I did choose Column>t test but graph pad for some reasons averages all my gene values for each group giving me one value/group and calculate the overall p-value. However, I want to see the significance of each gene alone between the two groups?
Thanks
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Dear Reema,
Firstly, let me tel the difference between paired-t-test and unpaired t-test.
Paired test is applied when you have one group, with before and after readings like, you have 6 animals and you want compare blood glucose values of these animals before the drug treatment and after drug treatment. here you are using same 5 animals, with 2 different readings as before and after, it is called paired comparison and hence we apply paired-t-test.
On the other hand, in unpaired-t-test, if you 6 animals as without treatment and 6 animals as drug treatment (total 12 animals) and you want compare the blood glucose values of these two different groups, we apply up-paired t-test.
As par as my understanding, in your study you are looking at comparing the gene expressions (5 genes) of control, and treatment (2 different groups), and in these scenario you have to apply un-paired t-test.
You have to compare the individual gene expression separately between the two groups.
If Raw Data Available
In Graph-Pad, go to Column option>choose graph>create>enter the group titles in column1 and column 2> enter the raw data for gene 1 for both the columns>analyze>t-test (and Non-parametric)>Unpaired T test>two-tailed>95%>4 significant digits>create a table>OK.
If you have only Mean, SD/SEM, and N
If you dont have raw data and have only mean and SD/SEM only
Grouped option>choose graph>select Mean, SD, N>create>enter the group titles in column1 and column 2> enter the Mean, SD, N gene 1 for both the columns>analyze>t-test (and Non-parametric)>Unpaired T test>two-tailed>95%>4 significant digits>create a table>OK.
Repeat the same for remaining 4 genes separately.
Regards
Vishwanatha
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Stem cells can be effectively differentiated into neurons. But why is the final neuron number very less?
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Kou Hayakawa
Dr. Steingrimur Stefansson is surely not miserable because he is still working in the lab, getting his hands wet and learning new things unlike Dr. Kou Hayakawa.
Ignoring your "true chemical results" (HepG2 fucoidan) that you trot out in all your answers does not keep me awake at nights.
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Hi Everyone,
I'm working on a self assembling peptidic scaffold which was designed for differentiation of stem cell into neurons. I have coated my plate surface area with 0.1 % of my peptide and seed some stem cell like bMSC and after on day I see some clustered structures on my plate while the CTR are okay. I was wondering what issue can be considered in formation of such aggregated and clustered structure?
Thank you in advaned
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Thanks you Mr Chahal, actually I did not used RGD and I think it is the problem in which the cells don't attach to the surface.
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Update: Cells are growing fine in our lab now!
I'm the only person in my lab starting on iPSc culture (we received a vial from another lab and all I have is some the medium they use for culture) and I'd like some tips and help. I have human iPS cells, and I culture in mTeSR1 on matrigel coated plates (so feeder free), replacing media daily. I try to remove what I think is differentiation by marking on the plate while looking through a microcscope. One cell line shows nice colonies like I saw on suppliers webpages (Like Stemcell or Invitrogen), the other I'm not so sure... 
I've attached some pictures, can someone tell me if this is what it's supposed to look like? And can give someone tips on how to passage? I've passaged one with gently scraping, like i saw mentioned in literature and tried picking the colonies with a p1000 tip (but didn't know if I actually picked up a colony or not, so I tried scraping). Maybe i just need a bit more patience :) 
While awesome to start things up, it's frustrating having no one to ask if things are supposed to be this way :)
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"When you plate the cells in low densities use ROCK inhibitor to prevent cell differentiation and/or death. With higher densities you should be fine without any. Cell passaging can be done using TrypLE, Accutase or dPBS + EDTA, I would let them grow till around 70%, more and they start to differentiate."
Sorry, but this answer makes no sense. Density is not the problem that is resolved by ROCK inhibitor. Clump size is the determinant. You can have a very dense concentration of single cells and they all will die, while larger clumps will survive.
The same is true for susceptibility to differentiation. Percentage of confluency is not the problem. The size and age of the individual colonies is what determines the likelihood of differentiation.
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Dear all,
I was wondering since stem cells are differentiated to various cell types some of the loading controls (eg ACTIN, TUBULIN and GAPDH expression) may vary during differentiation. Could Ponceau staining (detected on blot) serve as a loading control ? If yes, how could it be used to act as a loading control (quantitatively)? I would like to know how could we quantify the loading controls (meaning that quantify all the bands in a given lane and then take relative values??) OR some other method.
Thank you very much!
Kind Regards,
Faisal
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Hi everyone,
I did Ponceau Red on my samples of NSCs differentiating to two lineages (glial and neuronal), hoping to avoid the loading controls problem as mentioned above. But I am not any smarter after doing this. If you look at the attached picture bellow you can see clear differences in the patterns of the staining between stem cells (first 3 lanes), glial (different time points, next 9 lanes) and neuronal (different time points 9 lanes at the end). I loaded the same amount of protein (20ug) but this clearly cannot be used to standardize :(
If anyone had similar problem and/or has any ideas how to do the loading control for these samples would be greatly appreciated. I am running out of options :)
Thanks in advance.
Barbora
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I haven't used hESC before. I have a lentivirus construct that I've used in differentiated cells to knockdown the same protein.
What I should do differently in hESC compared to differentiated cells? Media, duration of treatment etc.? Should I look for background differentiation of stem cells?
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Ι don't se any reason why a shRNA not to be working in ESC. By the way is the construct working in other cell types? What is the promoter driving expression
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Dear researchers,
Can you please tell me the mechanisms or the pathways of mesenchymal stem cells differentiation into nucleus pulposus-like cells? Please explain me in details.
Eagerly waiting for the answers to my question.
Thank you very much!
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Transforming growth factor-β (TGF-β), insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF) are all upregulated when MSCs differentiate into NP-like. However, it appears that the GFs are not a sufficient stimulus toward differentiation. What Yamamoto et al., Tao et al. and Ball et al. suggest it is the key trigger to differentiation into NP-like is direct co-culture:
From Tao et al.: "Co-culture of MSCs with direct contact showed substantial increase in gene expression of Sox-9, aggrecan and type collagen, indicating that direct contact is essential for MSC differentiation to NP cells. MSCs co-culture without contact did not show any significant changes in gene expression. It is concluded that the cells with which MSCs are co-cultured determine their fate and that co-culture of MSCs with NP cells with direct contact produces NP cells differentiated from the stem cells. Moreover, like NP cells, MSCs presented a ring-like appearance."
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Differentiation of stem cell or zygote
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Hello Khalid.
Genetic expression and environmental cues direct cell differentiation. Gene expression is regulated by activation of specific genes according to autonomous and non-autonomous signaling pathways. For example, as early as 2-cell embryonic stage, cell interactions would be environmental ques including oviductal fluid while pluripotent transcription factors divided within the fertilized embryo will drive totipotency of the blastomeres, becoming decreased as development proceed. More external signals released and cell receptors in developing embryos would be involved to instruct embryonic cells to differentiate into specific cells. As the embryo develops further into a complex cell layers of embryonic structure and vascular system established within the embryo, other emryonic cell niches such as physical forces, body fluids, nutrients, gases, and hormones would also affect cell differentiation. Thus, cell differentiation is a complicated results of combinatorial gene expressions within the embryo.
Regards.
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Differential gene/protein expression profiles on same cell line always shows varied results, why does this happen?
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In last decade, accumulated evidence reviewed that dopamine and its receptor expression in human lymphocyte. My previous study found opioid receptor binding in blood lymphocyte of opioid addicts were down-regulated when compared with healthy control using radio-ligan binding assay. My current study in amniotic fluid stem cell-differentiated to dopaminergic neurons,  I accidentally found that blood lymphocytes expressed tyrosine hydroxylase (when I used blood lymphocyte as a negative control for TH expression but they are positive to TH by western blotting). It is interesting that blood lymphocyte expressed the rate limiting enzyme for dopamine synthesis. So, It is possible that blood lymphocyte may produce its own dopamine. Do you know what is the possible role of the dopamine system in opioid receptors in peripheral blood lymphocyte? Is its regulation of dopamine synthesis occur in peripheral lymphocyte similar to central nervous system? Thanks for sharing and discussion.
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Dear Prof. Sang Ho Lee
Thanks for your insightful discussion. You well described by raised how the brain and neuroendocrine cells have soft-wired synapses by secreting components to give signals to another system. This clarifies the possible way to have immunological synapse among brain, central and peripheral immune cells.
Thanks for your kindness
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Hi
I used two cell lines hESC cutured in E8 medium feeder-free condition.When I convert into naive state use protocal by 3iL or NHSM media,I find the colonies shows continuous apoptosis and cannot passage even though forming a little dense colonies as picture.First I think the high concentration of every inhibitor harms to cells in consideration of individual cell line,but when I reduce the dose,it does not work.
Does anyone have similar experience or can give me advices?
Many thanks.
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Hi,
Have you figure out the way to convert the cells to naive state without using MEF feeders? I am now trying to use the 5iLAF in MEF-conditioned medium. Some domed colonies appeared, but very small along with massive of cell death.
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in which ways can are searcher control stem cell differentiation?( in vitro)
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This is a very imprecise question.  If you are talking about embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) then there are many protocols available to differentiate these cells down different lineages.  By removing LIF and feeder cells ES cells will differentiate spontaneously and randomly, but if you culture them under certain conditions and provide certain cocktails of factors you can control the lineage.  So investigators have derived ways to make neurons, or cardiomyocytes, or renal epithelial cells.
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I know that study on development and differentiate of stem cells are very useful for therapy purpose and other useful purpose and it can achieve by microscopy on living cells.
but what is purpose of study on fixed and cleared stem cells ( as they are dead and fixed cell and have no differentiate and developing activity)? what information and data can we obtain from this study?
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Hello, Fariborz.
With a simple visual inspection of stem cell differentiation, you can tell only that my stem cells are differentiating in responses to inductive signal(s) probably due to the different cell shapes or motility. However, you cannot tell what proportion of cells respond? or are those differentiating cells are functionally mature of immature? or what cell lineages are present in the mixed cell population induced by the signal(s)? To see cells more closely, cell clearing with many detergents cocktail may be necessary to allow the probes to permeate or to infiltrate . All these questions can be answered by analyzing all cells present in the culture. The results obtained from the analyses further define technical improvements to generate more efficient ways of cell differentiation in culture. This is why we fix cells to preserve the moment of we collected from culture. Then we could look at the molecular and cellular components in detail with a great deal of probes available. Often cells are collected as live for further analyses for biochemical and molecular properties to understand principle of stem cell biology that is applicable to cell replacement therapy.
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Can we use  Alzarin Red S 2% solution for staining differentiated stem cells ( osteoblasts) stored in fridge up to 9 months ? Is it still stable and valid to use knowing that it is wrapped in foil and stored in dark in fridge 
Hope any one could help me with this 
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If your samples are not that too valuable you can try of course... Still I would not recommend it. If there is no staining you will not be sure if the solution caused the problem or the sample does acutally not contain any calcium.
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Is there a way I can differentiate peripheral human memory B-cells into Ig secreting cells (ISC) via T-independent pathway? I understand in vivo this can happen through LPS stimulation in presence of BAFF/APRIL. But I wanna know if we can do this ex vivo using humen memory B-cells from PBMCS.
Thanks :)
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Hi Subhranshu,
What cell concentration you use for culture and the total IgG concentration you received of these culture supernatants? Also it would be great if you can share your protocol?
 What percentage of your CD19+B cells turned  into Cd20-ve Plasma cells?
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I wanted to know if we have to be careful of this difference while ordering growth factors to use in media for differentiation from human embryonic stem cells to endoderm/ectoderm lineages. Functionally has anyone observed any difference?This link below made me wonder...
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Non-glycosylated growth factor might not give you the full potency but most of the time it will not affect your results dramatically. Say like VEGF the glycosylated have longer half life but some studies show the acute effect might not be different from glycosylated one. Protein yield from eukaryote cell is low and harder to purify but CHO is a eukaryotic line which should provide you reasonable glycosylation. 
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If anyone is related to the organic chemistry of stem cells, does anyone know of the most important reactions involved in stem cell differentiation?
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Hi Xavier, I think you asking the wrong question here.
All nucleated cells, stem cells included, have the same organic chemistry of anabolism, catabolism, protein turnover, etc, etc.
The question should be how stem cells can be initiators of organ/tissue repair or cancer.
That is not dependent on organic chemistry, but where the stem cell is and how it is influenced by neighboring cells, malignant or otherwise.   
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Dear All
I am working on Neural stem cells based In-vitro Differentiation  Assay Can any body of you suggest me the best Coating material of Cover slip for Differentiation of Neural stem cell of Hippocampus into Functional Neurons. At the moment I am using ECM gel for this but I am not satisfied with them
Thanks
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They dont really like to be on a glass surface! If your aim is staining, I would suggest you to use the ibidi slides. They are compatible for microscope!
Matrigel is widely used but lot differs a lot! I would go with pol/laminin co-coating, they are very good for long term culturing and i would spike laminin every 10days into the culture media.
Good luck
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I've read a number of protocols on this and several people's comments, but try as we might our lab is struggling to make this a success; whether we are culturing to differentiate macrophages (conditioned L929 media) or dendritic cells (GM-CSF), we're left with virtually zero viable cells at the end. I suspect that the hematopoetic progenitors are not surviving the process.
After I collect the bone marrow, I wash them once with complete DMEM and then resuspend in 90% FBS + 10% DMSO.  Aliquot the cells (10^7/tube) into cryotubes and then into rate-controlled freezing container overnight and transferred to liquid N2 the next day.  For recovery, we put the frozen tube into 37 deg waterbath until *almost* thawed, transfer the cell slurry into pre-warmed complete media (~20-30 mL), centrifuge to remove DMSO, and resuspend in differentiation media. 
The only things I can think of that might still be affecting our results is 1) the concentration of cells/freezing media may be too high or too low, 2) thawing is too quick (although I thought that was the point), 3) handling is too rough; the cells need a rest period before differentiation.
But truthfully, I have no idea. By all accounts I've heard, we should at least be getting SOMETHING. When we prepare macrophages or DCs from fresh bone marrow, all is well and our yield is excellent. 
Does anybody have any thoughts? What the heck are we missing here?
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I have noticed that following retrieval from cryopreservation, the hESCs that we are using experience a cell "crisi" following 3 passages. Although I have heard anecdotal evidence of this being a common phenomena I was wondering if anybody knew if there had been any research carried out as to why? 
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the problem only occurs after passage 3, cryopreservation  in N2 seems to reset it. ROCKi is used following each passage. We have noticed some differentiated cells in the culture, but these largely remain behind following passage. We will be performing some flow analysis soon. Thank-you for everybody's input, it is greatly appreciated- as would any other feedback 
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Hello to all, recently I used the hanging drop methods to obtain murine embryonic bodies. Do the embryonic stem cells need to be selected? If not, will the fibroblast effect the formation of the embryonic bodies? Besides, does the passage number effect its differentiation?
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Hi Kejun,
Your EBs should still form nicely but one would expect 'contaminating' EBs to have an effect on the subsequent differentiation of your mESCs.
If you are worried about the fibroblasts you can try to deplete them, seed your plates for 10-20 minutes, tap them to dislodge your ESCs and replate those. Fibroblasts tend to adhere much faster than ESCs allowing you to deplete them this way. There are other methods (but more costly), e.g. miltenyi has magnetic beads to remove fibroblasts.
Will passage number affect differentiation: I would say definitely yes. In my experience EBs from a late passage mESC actually formed beating cardiomyocytes easier. This is likely because of my culture conditions, the FBS used etc resulting the skewing of my mESCs toward mesoderm differentiation. 
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Hi everyone,
I'm doing differentiation of the stem cells. We plate the cells on matrigel but need to detect in the final stage with IF. I've tried once stain directly in the plate but unfortunately I couldn't get a good image as the reflection of the plates. So I'm wondering if I put a slide in the well and plate with matrigel, will it affect the differentiation efficiency? Has someone done this before?
Thanks.
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Hi Oscar,
Yes you can put cover slips in your plates coated with matrigel, so when you wish to perform IF you can remove the cover slip.
Alternatively better option is to use chambered slides. So you can grow cells in these chambered slides and when you need to observe under the microscope you can remove the chamber. 
And do not worry of efficiency of differentiation.
All the best.
Regards,
Prasad.
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Publications on the use of differentiated human iPSCs-neurons contain characterisation information about the different stages of differentiation (iPSC-NPC-neurons). What information is needed for this? PCR? FACS? IF? how much needs to be done at each stage? 3 repeats per clone, etc?
Any guidance would be very helpful! Thanks! 
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Hi, 
The transcriptional analysis by RNA-seq would be the best.
The analysis should be carried out at the level of mRNA (RT-qPCR) and proteins (WB, ICC, FACS or ELISA).
Electrophysiological characteristics of mature iPSC-derived neurons should be performed.
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I am currently trying various differentiation protocols, but I have not had much luck when it comes to positive results. I know that the yield is generally low, but does anyone have a basic protocol that has worked well for them? Any common pitfalls that I should avoid when dealing with this lineage?
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i want to culture neural stem cells from E14 mouse cortex. which neural stem cell culture gives the best result? what growth medium components are to be used? 
for serum free media supplements? what do you recommend to use? 
would using only GS21 be enough? or should i use b27 and N2 for better results? and also which vendor do you recommend to buy the supplements from? 
thanks in advance
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Hello,
We use Esc medium (with KSR), Neurobasal medium, N2 and B27 supp. to make dopaminergic neurons, but must be used in different times of differentiation.
I recommend you follow a article that alredy did this culture and diffrentiation (oligodendrocytes) from mouse cells.
You will spend less time following a protocol already established.
Regards,
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I'm dealing with human induced pluripotent stem cells culture using Matrigel or vitronectin XF coated plates. I passage the cells as small clusters and used ROCK inhibitor with mTeSR1 medium for the first two days of each passage. It has been fine for the past but recently, cells would detach and die. At first I through it was because they were too crowded and do the passage before it was too dense. It seemed to work. Then last time during passaging, I broke cell clusters into mostly single cells and supplemented with ROCKi all the way during culture. They grown normally for the first 5 days, then detach before reaching optimal confluency. I checked literatures but find nothing about long term ROCKi usage would cause detachment. Please help me find the reason.
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Hello,
Yes, rock is used only in passage or defrost (first 24 hours). After 24 hours you must changing meium take off it. Normally diluated in DMSO that can differentiate your iPSC too.
I agree, maybe the problem is with your sample of the medium or matrix.
We work with iPCS in clamps and single cells, both works good.
In our lab we use E8, mTeSr1, L7 médium, gtx, matrigel, vitronectin, L7 matrix...
When we have problem first we check reagents (see with others researchers if has same problem. If yes, use new samples that you are using same) and incubator (water, CO2, O2, temperature...)
To dissossiate cells we use:
- Vensene, Accutase , Tryple, Relesr = passage in single cells
- Versene, Relesr, Gentle = passage in clumps
- Dispase, Collagenase IV = to make Embryoid Bodies
Regards
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My mouse embryonic stem cell line are not working. They are not able to attach to the surface of coated dishes ( I have tried both Geltrex and 0.5% gelatin) remain in suspension. I am using ESGRO LIF. Please help 
Media composition =  KO-DMEM+15% FBS+1xGluatmax+1xNEAA+BME+PS 
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You should start by checking your FBS.  Always use ES-qualified FBS.  We have had batched of regular FBS that do not promote adhesion of ES cells.  Different lots of ES-qualified FBS still give variability, but at least they are screened to confirm adhesion.
You should also check how the ESCs were cultured before they were frozen.  If they were on MEF feeder cells, you will want to thaw them on feeders.  If they were adapted to feeder-free, confirm the matrix. There will be less stress to maintain the same conditions they were adapted to before frozen.
Confirm your final concentration of b-mercapto.  I have had techs use the wrong bottle at the wrong dilution and wonder why the cells are not attaching.
ES cell culture is less forgiving than other cell cultures.  If you are still having problems, find a PI or mentor for more hands-on training and practice.
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I am differentiating murine muscle-derived stem cells into neural lineages according to standard protocols. I coated the dishes/wells with Ply-L-ornithine and Laminin. After plating the cells, they attach really fast and nicely. However, from the second day on, they start to detach and form floating neurospheres. Any idea why it is happening like this? Any suggestion for a protocol and coating method which may work?
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Hey, I have the same problem right now. Did you find a solution for it ? If so please share it with me.
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Topics for making a good proposal on cardiomyocyte differentation. ? Can anyone help? to be more specific iPSCs to Cardiomyocytes differentation
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thanks a lot
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I want to Know how stem cell differentiation occur for general and depend on what happen either in vivo or in vitro and if  this mechanism  differ from one cell type to another or not.
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Does anyone know a way of differentiating E14 stem cell into cardiomyocytes without using hanging drop method? Easy 6 well culturing and harvesting for biochemical assays.
Thanks :)
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Hi, I did the differentiation of E14tg2a cells via embryoid body formation on low adherence plates/wells in absence of Lif. Within one or two weeks some beating bodies will form which can be plated or harvested for analysis. 
Kind regards,
T.Redmer
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Hi, I have a project about using the micropatterns substrate to see the hMSCs behavior and the differentiation fate. the project goals is to find out whether the micropatterning can lead the direct differentiation of MSCs to become chondrocyte lineage. So are there any specifications of the micropatterns especially to enhance and direct the hMSCs becomes chondrocyte lineage?some references or research articles link related to this project may be helpful. Thank you. 
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Hi, you can try my old department, they have publications on micro/nano patterning on stem cells at glasgow university tissue engineering. Check Mathis Rhiele, Matt Dalby publications they have extensive studies on stem cells and surfaces.
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Hi everyone, I'm culturing mES recently, and I have found that my mES differentiate quite quickly, often on the second passage off MEF feeder. Could anyone with experience in preventing mES differentiation?
PS:I use MEF feeder plus 0.2% gelatin to culture mES. The medium used is Gibco's KnockOut DMEM, supplemented with Gibco's FBS, P/S,NEE amino acids, β-ME and LIF.
And the pictures of my mES are also enclosed
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Hi Sharif,
I'll try as what you told me, thank you very much!
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When my lab isolated and characterized cells from bone marrow, we found about 30 different cell types, i.e., totipotent stem cells, pluripotent stem cells, germ layer lineage stem cells, ectodermal stem cells, mesodermal stem cells, endodermal stem cells, hematopoietic stem cells, hematopoietic progenitor cells, mesenchymal stem cells (Caplan’s), stromal vascular cells, endothelial progenitor cells, endothelial cells, endosteal cells, osteogenic progenitor cells, osteoblasts, chondrogenic progenitor cells, chondroblasts, adipoblasts, unilocular adipocytes, etc. Which cell population are you using for your studies?
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This may not be an answer to your question about the nature of the cells but it is an important point in development of cell cultuer media using human albumin as a constituent and carrier molecule.  I understand that American companies that factionate human plasma use a mix of tryptophan and octanoic acid to stabilise albumin during the 60C heat pasteurisation step of manufacture.  CSL Behring in Australia uses just octanoic acid but at double the concentration and this results in a much higher level of this excipient that adversely affects mesenchymal cells in culture.  The link below should take you to the paper.
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I was looking for an article which has proven to be induced pluripotent stem cells are a better stem cell source for neural differentiaion but i could find nothing.
if a cell is less differentiated ( pluripotent Vs. multipotent or iPSC Vs. MSC ), does it mean that cell differentiate better to any differentiatied cell?
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We are using iPSC to differentiate into neural cells in our lab. We reprogrammed skin fibroblast into iPSC. 
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Dear all, while SB431542 is established to block TGFbeta and LDN193189 to inhibit the BMP pathway, I was wondering if anyone has seen the combined effect of the two in neural differentiation of mouse embryonic stem cells? Their combined effect causes neuron production in human ESCs but I couldn't find any literature for mESCs. (http://www.nature.com/nbt/journal/v35/n2/full/nbt.3777.html)
Is SB431542 only used to maintain pluripotency and inhibit differentiation in the mESCs instead ? (https://www.ncbi.nlm.nih.gov/pubmed/24949833)
Would appreciate your experiences/ suggestions/ ideas.
Many thanks.
Pooja.
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Both GFP positive and GFP negative cell population decrese phosphorylation when you use LDN and SB together. I read this paper some time ago. Please see this paper and you may write to Author if you need. here is that " TGF-beta-superfamily signaling regulates embryonic stem cell heterogeneity: self-renewal as a dynamic and regulated equilibrium" published in stem cells January 2013. 
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Hello! I would like to ask about the quantification of sGAG after the chondrogenesis of mesenchymal stem cells.
My principal doubt is how to extract the sGAG to performs an assay with Dimethylmethylene Blue Assay (DMMB) because I have found protocols for that and they talk about the complex sGAG-DMMB but they do not mention how to obtain the sGAG in solution... Maybe with the application of the reagent the sGAG are realising to the medium as sGAG-DMMB complex directly after following the protocol?
Another issue, if I do Alcian blue/nuclear fast red stain. I cannot use them for the abovementioned assay right? I should have two pellets (one for the histology and one for the quantification)
Thanks a lot,
Sergio.
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Alternatively, you could use an Alcian blue based assay. But be forewarned they stopped making Alcian blue back in 1979 due to its highly carcinogenic activities during manufacture (large number of employees making stain were getting cancers). So any Alcain blue purchased after 1979 is not anywhere near to being 100% stain, it is mostly fillers. Alcian blue assay protocols can be found in:
Young HE, Dalley BK, Markwald RR. Glycoconjugates in normal wound tissue matrices during the initiation phase of limb regeneration in adult Ambystoma. Anatomical Record, 223:231-241, 1989.
Young HE, Young VE, Caplan AI. Comparison of fixatives for maximal retention of  glycoconjugates for autoradiography, including use of sodium sulfate to release unincorporated radiolabeled [35S]sulfate. Journal of Histochemistry and Cytochemistry, 37:223-228, 1989.
Young HE, Carrino DA, Caplan AI. Histochemical analysis of newly synthesized and resident sulfated glycosaminoglycans during musculogenesis in the embryonic chick leg. Journal of Morphology, 201:85-103, 1989.
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Is my first time working with stem cells and I don't what kind of stem cells are better for this. 
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It would be helpful if you specify which kind of neurones do you want to obtain. There are plenty of protocols published and some commercial kits that can be useful (I would recommend you to better find a protocol than a commercial kit since they are quite expensive). 
And also what kind of stem cells: if you want to start with pluripotent stem cells (embryonic or iPS) or if you're starting directly with neural stem cells, if they are human or from any other source, etc. Then I guess people might give you more specific answer.
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It's known that Matrigel, in general, is used for culturing pluripotent stem cells. I wonder if Matrigel does show any adverse effect on mesenchymal stem cells, such as directing them towards a spesific cell type, when they cultered on Matrigel instead of gelatin or collagen.
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Hi Burak,
i use the Growth Factors Reduced Matrigel in my  osteogenic differentiation of hMSCs and i don't see any specific background stimulation by the coating itself. Hope it helps. 
Regards,
Laura
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Hi, I have been trying to induce neural induction in my mESC lines by using -DMEM-F12 + N2+B27+NEAA+ bFGF. Although I started getting rosette formation from day5/6 onward, and mature neural marker expression from day7 onward, I also had lots of other lineage (mesodermal/ endodermal) contamination. I performed this NI for a period of 14 days, changing media everyday.
To counter the other lineage contamination, I started with a different protocol using small molecule inhibitors using the following media cocktail- Neurobasal media+DMEM-F12+NEAA+Glutamax+beta-mercaptoethanol+insulin+ B27+N2+bFGF+SB+LDN. Although this protocol has worked well in the hands of another researcher using mESC HM1, it is not working at all with my lab's established mESC lines. Even until the day 8 of N.induction, I can't morphologically see any neural lineage appearance. I just came across this publication: http://www.sciencedirect.com/science/article/pii/S0898656814001971 which discusses the role of SB in inhibiting differentiation and maintaining pluripotency of mESCs. 
I am quite confused at this point, and would highly appreciate if someone could shed some light on these issues I am facing. I have cells in culture in both conditions right now, I am tempted to take out SB in some wells and see the effect, but it would be great to get some advice before I go manipulating the protocols.
Many thanks.
Regards,
Pooja.
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Hello Pooja,
Truly speaking, you have made your second protocol quite complicated. First protocol is good but I will suggest some changes. N2 supplement is known to favour the expression of neural genes and inhibits the growth of non-neuronal cells in the culture (or any undifferentiated cells). You can use N2 supplement (1%), add G5 supplement with it to increase the efficacy. Use neurobasal media and there is no need to use DMEM+HAM F12 , NBM is sufficient for this purpose. Please remove excessive things from your supplements like insulin, BME etc. as they can support growth of nonspecific cells. Use 2% KSR to replace FBS. Please give your updates about the same.
Hope it helps !
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I want to over-express various genes in human ES and iPS cells, mainly snoRNAs and the expression to be stable both in the pluripotent cells and when I differentiate them into the 3 embryonic germ layers. Which vector, promoter, transfection system and protocol would you advise me? I am currently growing the cells as mononlayers in feeder-free conditions (mTESR1&Matrigel). Thanks!
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I used lentivirus too and it always worked for me. You can find also an protocol for  transfection on my side. Look up for:
Real-time tracking of fusion between adipose-tissue derived stem cells and myocytes using a dual fluorescent protein reporter vector (pLVX-Puro-HS-GR)
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Hello,
Recently, I tried differentiate MSC from hESC or hIPSC , but i was failed.
There are many published papers described briefly about  MSC differentiation from hESC or hIPSC.
I just followed some published methods.
"PLOS ONE, January 2013 | Volume 8 | Issue 1 | e54524" this Paper stated that 
"Undifferentiated hESCs were grown to reach 70% confluence.
mTeSR1 was then replaced with MP media (DMEM low glucose
with 20% fetal bovine serum (FBS), and 1% penicillin/streptomycin).
Differentiation was allowed to proceed for 6 days in this
media. Cells were then passaged using dispase (1 mg/ml) and
plated on matrigel coated plates for a further six days of culture.
Media was changed every 2–3 days."
but, When I did experiment, after adding MP media the cell growth was fast, and I found cells were floated and died. so next time i subcultured cells within 2~3 days on Matrigel coated dish. then, I found that cell attachment was not good and cell condition was bad.
My question is "When should I subculture cells during MSC differentiation and which coating material will be the best? And If possible could you suggest another protocol what can meet my desire?
Thanks in advance......
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We developed a protocol for robust production of MSC from iPSC that will be published soon in npj Regenerative Medicine. We found that during early passages, the inclusion of ROCK inhibitor dramatically enhances survival and replating after passage. Over the course of subsequent passages, the cells can be weaned off ROCKi.  
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I have cultured MSC from Mouse bone marrow.
These pictures are passage 5 MSC.
I think 2 pictures very different cell shape .
I want you think about 2 pictures.
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The A shows MSC, but as others also mentioned you need to split them ASAP. However, B could be MSC but either after split/seeding or they are not feeling good :D. 
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I would like to transfect human embryonic stem cell differentiated cardiomyocytes with GFP plasmid. Based on the literature, it does seems like, these cells are relatively difficult to transfect. I am looking for any such suggestions or experiences with transfecting hESC-CM, and also suggestions regarding which transfection reagent suits best for transfecting these cells, would be truly helpful. 
Many thanks for your suggestions or advice in advance, Reena 
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Thank you everyone!! 
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At the moment, I am trying to differentiate mouse iPSCs to the neural lineage. So far I've carried out neural induction (N2+ B27+Dorsomorphin, SB43..., CHIR and Noggin) for 4 days and replated them onto polyornithine-laminin coated plates for culture in Neural Expansion (FGF + ITS + B27+ N2). I get a mix of different "neural" cell types (2 main ones. see pics attached) some of which I'm unfamiliar with. My goal is to obtain first neural stem cells based on their morphology, expand and then subject them to differentiation. I would much appreciate your input.
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Hello Ismael,
I doubt that Neural progenitor cells and neural stem cells can be differentiated morphologically. And I don't think that there is any difference in neural progenitor cells and neural stem cells. There is a great deal of ambiguity in defining these two types and people often use these terms misnomerly. According to me they both are same as I have read literature on same. 
Coming to morphological difference, as per my experience you can't differentiate these cells on the basis of morphology. Go for specific antibody staining for that. That will be more reliable approach.
Hope it helps !
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Can anyone who has experience with neuronal differentiation suggest for a replacement to human recombinant Noggin? Any other signaling molecule with same functions? 
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Hello
i agree withe Nina Pipalia , LDN-193189 its good 
Look at the PDF here will help you
Good Luck 
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We have an EDC crosslinked collagen based material and are performing a degradation assay to understand the byproducts released and quantify them. Is there an assay that exists to detect EDC in any form (residual, unreacted, partially reacted, fully reacted) as the material degrades or upon initial hydration? I understand there is a free Chlorine when EDC is unreacted that is no longer there upon successful crosslinking. Our initial thought is to focus on the presence of Chlorine as an indication of residual EDC. Any thoughts are appreciated!
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My pleasure. Hope it works for you and good luck for your submission. 
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In my study, I am going to observe the cytokine production level in PMA differentiated THP-1 cell after drug treatment.
1. Does PMA itself stimulate cytokine production?
2. if yes, how can i fix this?
thanks in advance.
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It is a positive stimulator of TNF-alpha
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I am working at the problem of mammalian development and stem cell differentiation. And wonderingly how can we define different cell types, and how many cell types in a specie. Any one know where can I find the answers?
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Dear Jinshi,
Several articles have dealt with this interesting question:
McCarthy* MC, Enquist BJ. Organismal size, metabolism and the evolution of complexity in metazoans. Evol Ecol Res 2005;7:681–96.
Valentine JW, Collins AG, Meyer CP. Morphological complexity increase in metazoans. Paleobiology 1994;20:131–142. doi:10.1017/S0094837300012641.
Carroll SB. Chance and necessity: the evolution of morphological complexity and diversity. Nature 2001;409:1102–9. doi:10.1038/35059227.
To determine how many cell types are in a tissue or organism is more complicated.  The main issue is how you define cell types? Morphology, transcriptomics (as Mohamad said), proteomics, etc. All these methods allow you to identify  cell sub-populations but this may be not cell types, which adds a notion of differentiation pathway. And if different cell types should have different profiles, the reverse is not true.
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Right now I have a protocol from Lifeline Cell Tech. that requires one to make chondrogenic microbeads from MSCs using an alginate solution and their proprietary media. However, I have seen several techniques that simply consist of treating a pellet with lab-made or proprietary differentiation media. I am wondering what protocols the community uses, and if you've tried several different protocols/products, what do you find are the strengths of the protocols/products you use.
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Dear Carl,
In my hand, the easisest way to do is to make pellets with you MSC in a 15 ml conical falcon tube. Following centrifugation, simply detach the pellet from the bottom of the tube. Remove the medium and add the differentiation medium (Dulbecco’s Modified Eagle’s Medium, High (4.5 g/l) Glucose supplemented with 10-7 M dexamethasone,
50 mg/mL ascorbate, 50 mg/mL Insulin-transferrin sodium selenite, 100 mg/mL sodium pyruvate, 40 mg/mL proline, 2 mM L-glutamine, and 10 ng/mL TGF-b. After 21 days, you will have nice chondrocytes.
Best regards,
Thomas
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  • Why c2c12 myoblasts cells do not differentiate by adding 2% Horse serum?
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Thank you very much for your precious help.
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Q1. I am planning to use an ALP kit to detect the mES cells pluripotent/ differentiation status. Which would be the better (ALP) kit for this purpose?
Q2. I generally passage mES cells every 2-3 days. But some kit says 4-5 days (crucial for ALP activity). The detection days (4-5) are very strict? If grow the cells for 5 days, the cells were not looking good., may be started differentiated (EB?).  
(I used 2i+LIF medium with supplements)
Q3. Does anybody having experience of using- Vector Blue Alkaline Phosphatase Substrate Kit III (from Vector Laboratories) for mES cells?
Thanks in advance!
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Hi Ramanan,
I work with human iPSC and I guess 4-5 days could be sometimes too long for ESC which might lead to differentiation. Proliferation rate might differ from cell line to cell line (sometimes even for clonal lines), so I would recommend to passage your cells as you normally do (AP staining would not work when the cells are differentiated, right :)
I tried live AP staining on my iPSC, which worked nicely. One advantage is that you do not have to fix the cells and they survive the staining with no differentiation or some other side effect. Have a look at the link if that might be of interest to you.
Good luck.
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Hi everybody,
I want to establish a protocoll for isolating and culturing primary rat neurons from E16-18 rats. The culture does not have to be neurons-only, but should be reproducible in terms of composition of cell types. The simpler the protocoll is, the better.
The cells are later going to be used to establish patch-clamp experiments.
I am specific about E16-18 prenatal rats because we are isolating DRG-neurons from those animals and I would like to use the same animals for my brain-cell-isolation.
First I had the idea to isolate cerebellar granule neurons (found an easy protocoll for that), but it seems these are not yet developed until birth?
I am looking forward to your comments and ideas, thanks in advance!
Cheers,
Jörg
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Margaret, thank you also for your comments!
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Differentiation to osteoblast using C3H10T1/2 (MSC cell line)
The protocol that I have found making osteoblast uses DMEM, also using 10mM beta-glycerophosphate, 50ug/ml ascorbic acid (2~3days).
(Protocol)
1: DMEM (high glucose), 10% FBS, 1%P/S (growth media)  C3H10T1/2 cell 5x10^4 split in 6well.
2: when reaching 100% confluency in 6well, changed media every 2days with DMEM (high glucose), 10% FBS, 1% P/S+ 10mM beta-glycerophosphate, 50ug/ml ascorbic acid (differentiation media) .
3: on the 7th day, collected cell with Trizol and looked for ALP, Runx2, Osteocalcin, Osterix (osteoblast marker) with qPCR.
4: Results were no increase in ALP, Runx2, Ostepcalcin, Osterix (mRNA level). This can be inferred as nothing has differentiated to osteoblast.
I have looked numerous journals related to this but nothing was different from my protocol. (Some said to coat the plate with 0.2% gelatin but still didn’t work. Also using MEM-alpha didn’t work)
My questions are
          1: is there any specific media when differentiating?
          2: or should I change the concentrations of my media that I am currently using. (Seen journals using dexamethasone but all the concentrations were different)
          3: Would coating plate matter?
          4: I have differentiated for 7 days but would I need more time? (ALP should be seen in 7days anyways, but other suggestions would be nice)
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I have differenitated EC3T3's, so not sure how similar these cell lines are but your media sounds find, you don't actually need to add BMP's, most of these MSC cell lines just need ascorbic acid and beta glycophosphate.  However, 7 days is not long enough at least for the 3T3's.  Alk phos begins to appear at day 12-15 and alizarian red staining shows up at day15-17.  By qPCR I've tested differentatiated cell lines by 7-9 days but that was with differentiated Osteosarcoma cells which are different.  Go longer if you can and see if that makes a difference,  The only thing you might see is that after a while, the ascorbic acid will start for form these crystals in the media.  Don't be alarmed, the cells are not hurt by this.  I would set a side a couple of wells in a 24 well plate, differentiate those and instead of looking of gene expression first, stain then at day 5, 7, 9, 12, 15, ect and see if alk phos and alizarian red can be seen. This is cheaper and faster than qPCR.  Once you see signs of mineralization you can be sure you have differenitation and then you can work out the qPCR conditions and timing.
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I am culturing mESC RW4 cells and found that they start to differentiation after two passages.
For culturing, I used the knockout DMEM (Gibco), with 15% FBS (ES cell, Gibco), 2mM L-glutamine, 10mM Hepes, 0.1mM non-essential amino acid, 0.1mM beta-mercaptoethanol. The media is prepared freshly and used up to 1 week. Fresh LIF is added to the cell directly every time when changing media or splitting. I change the media everyday for mESC. And of course I used 0.2% gelatin for coating the plate and have MMC treated fibroblast as feeder cell for mESC.  
I attached the figure to show the morphologies of mESC at p1 (left figure) and p3 (right figure). mESC at p1 are undifferentiated with nice round shape. mESC at p3 have two types of cells mixed together. I think the round colonies are still undifferentiated. But those small round shining cells around the colonies look neither fibroblast nor mESC. To me, they are differentiated stem cell. I will use AP1 staining to confirm if they are really differentiated or not in the next step. 
I am so upset they are easily to differentiate. Due to the consideration that the FBS batch are not good, I have tried to change the FBS(optimized for ES cell) to knockout serum replacement (Gibco)  and at the same time change trypsin to accutase (Gibco). I have also asked another lab for borrowing the good FBS that have been tested by them. These changes did not help with maintaining the stem cell status.
 I am now desperate and would like to ask if anyone has experience the same situation and if any one could suggest any element or staff that I should be aware. Thanks a lot!!
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If you add fresh LIF all the time, feeders are redundant. You need either LIF or feeders. 
The lot of FBS could be a reason. We usually test several lots of FBS, and order 20 bottles of the best lot. 
If you have just started culturing mESC, there is one important step: breaking the cell clumps to a single cell suspension before plating. Usually, I lightly push the pipette to the bottom of a 15-ml tube, and pipette the cells (in 1-2ml suspension) for at least 20 times. 
You cannot handle hESC like this, but you must do it for mESCs. 
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Any suggestion that the amino acids will lead to any detrimental change on differentiation of monocytes in to macrophages in conjunction with recombinant M-CSF?
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Keep replenishing the medium with MCSF every two days. The key is pure monocytes to begin with. Alternatively a crude way is to put PBMCs in a flask with medium without serum or 0.1 % serum  for an hour at 37 in the incubator. Monocytes will stick to the flask and wash away non adherent cells and replace the medium with RPMI contain serum and MCSF. Your yield will be better. This is an old technique but still works. Good luck!
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Hello everybody.
I'm trying to culture and differentiate murine DPSC into neurons like cells, but the proliferation ratio is really low.
Moreover, present cells show different morphologies (no genetic analysis done yet).
Do you know any coating for the plate that would increase proliferation and differentiation into neurons like cells?
Thanks ;)
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Hello Francesco,
First, Proliferation and differentiation are two contradictory things...if u want to do efficient differentiation then u have to stop cell proliferation so that cells can differentiate properly. Second, coating is done to increase adherence and sometimes Fibronectin and laminin help in increased neural differentiation as they help in Na/K channel activity and hence increased survival and differentiation of neurons. 
As you cell proliferation is slow, so I will suggest you to standardise an optimum media first (preferably alpha-MEM works best for DPSCs+ good quality FBS), once you get desired proliferation upon standardised conditions then you can try differentiation without coating and even with coating parallely (FN and laminin). As per my experience with human DPSCs, I get very efficient neural differentiation even without coating.
Hope it helps !
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Hi, I am interested in doing pluripotency analysis of my mESCs, and want to include the earliest markers for differentiation. I do observe some form of differentiation in my colonies, but not sure as to which lineage they could belong to. I am looking for something that's probably before the neural lineage, but ahead of naive pluripotent nanog/oct4 expressing stage?
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Dear Pooja,
I would suggest
Gata-4 (yolk sac endoderm and later cardiac marker) , GATA-6 extraembryonic endoderm and visceral endoderm
Sox1-neuroectoderm
T (Brachiury) - early mesoderm
loss of SSEA-1 and nanog as general sighs of differentiation
alpha-fetoprotein - endoderm 
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I'm measuring protein expression across human embryonic stem (ES) cell (as control), differentiated ES cell and E15 fetal mouse tissue (presumably same stage as differentiated ES cell) by western blot. attempting to detect transcription factor which found to heterodimerize with 90% structurally identical analogue.  Result: No band for control (as expected), a band for differentiated cell, and 2 bands of mouse tissue (of different size). What could be reasonable explanation behind different protein size of differentiated ESC and mouse counterparts apart from ab specificity. using polyclonal rabbit IgG. 
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Why do you think it isn't antibody specificity?
Send a picture.
What about post translational modification/s?  Splice variants?  Shared epitopes?