Stem Cell Differentiation - Science topic

Dear Leticia,

What do you mean by differentiating? Did you checked using alcian blue staining for chondrogenic, alizarin red staining for osteogenic or oil red O staining for adipogenic differentiation? I supposed that what you meant was that your cells did not grow in monolayer, but tended to form clumps of several layers, was that so?. Stem Pro or other comercial serum free medium do not contain attachment factor, so you need to coat your flask with collagen or fibronectin, to allow your cells attach to the flask and grow nicely as monolayer. Different if you use serum or platelet lysate containing medium. Serum and platelet lysate contain attachment factor, so you do not need to coat the flask, but the cells will grow nicely as a monolayer

I used a kit to differentiate MSCs to osteogenesis and adipogenesis described in the enclosed reference. But chondrogenesis, I used method described in this paper. Cardiogenesis, I did not study. But you should find some publications on this too

A Simplified Method for the Aspiration of Bone Marrow from Patients Undergoing Hip and Knee Joint Replacement for Isolating Mesenchymal Stem Cells and In Vitro Chondrogenesis.

Juneja SC, Viswanathan S, Ganguly M, Veillette C.Bone Marrow Res. 2016;2016:3152065. doi: 10.1155/2016/3152065. Epub 2016 Feb 11.PMID: 27057356 Free PMC article.

Have you tried this protocol? I want to try it but I am worried about its reproducibility. I would love to know if you have been successful with it. Thanks!

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

Hi Suh Hee Cook

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!

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).

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.

Have you tried Wnt3a treatment? it is critical in induction and maintenance of pax7, while inhibiting myoD, myogenin in segmental mesoderm.

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.

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.

Thank you Ellen A G Chernoff

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.

I am learning to culture organoids. Many thanks for the updating information.

In simple words its the heterochromatin patterns which reflects cellular identity.

Tuj1 expressed only in diffrentiated cells. For stem cells yopu can use SSEA1 marker

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.

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.

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 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

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.

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.

"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.

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

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

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."

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.

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

That's almost philosophical question.

Define your goal (what exactly do you want to control, and why) and you'll probably be one step closer to the solution. :)

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.

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.

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. 

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.   

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

Hello

See these links 

Good luck

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 

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. 

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.

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.

Hello, kindly look at the link below

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,

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

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.

Hey, I have the same problem right now. Did you find a solution for it ? If so please share it with me.

thanks a lot

Hello

This a big question  you can see the links and book here to find answer

Good Luck  

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

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.

Hi Sharif,

I'll try as what you told me, thank you very much!

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.

We are using iPSC to differentiate into neural cells in our lab. We reprogrammed skin fibroblast into iPSC. 

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. 

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.

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.

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

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 !

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)

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.  

Thank you everyone!! 

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 !

Hello

i agree withe Nina Pipalia , LDN-193189 its good 

Look at the PDF here will help you

Good Luck 

My pleasure. Hope it works for you and good luck for your submission. 

It is a positive stimulator of TNF-alpha

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.

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

Thank you very much for your precious help.

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.

Margaret, thank you also for your comments!

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.

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. 

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!

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 !

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 

Why do you think it isn't antibody specificity?

Send a picture.

What about post translational modification/s?  Splice variants?  Shared epitopes?

Hello

Also these papers .

Good Luck