Questions related to Stem Cell Differentiation
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!
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.
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!
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
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 ;)
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.
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.
I want to develop a lab-made media for differentiation of ADSCs into Keratinocyte-like cells.
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!
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 :)
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?
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.
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?
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
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 :)
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!
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?
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!
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.
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?
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?
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
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.
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...
If anyone is related to the organic chemistry of stem cells, does anyone know of the most important reactions involved in stem cell differentiation?
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
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?
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?
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?
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?
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!
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?
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
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.
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
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?
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.
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.
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.
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
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?
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?
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.
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,
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.
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.
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!
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......
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
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.
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!
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.
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?
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.
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!
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!
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).
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)
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!!
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?
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?
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?
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.