Stem Cell Biology

Thanks a lot everyone.

Hi Meera, according to the posted site, 10 minutes trypsinization time is the norm. How long it will take depends on how fast your flask warms up. As suggested by previous comments, add warmed up trypsin. Then place flask into incubator for about three minutes, then remove it and quickly tap the sides. If the cells are just starting to come off, put it back for a further 1-2 minutes and repeat the process. How fast the cells come off depends on how fast you work. If you are too slow when visualising, trypsin cools down and activity reduces. However, the cells that came off would be still affected and then they may not attach upon seeding. One very important thing. When about 90 percent of the cells detached, quickly add the neutralising solution that stops the activity of trypsin. For most cells it is FCS, I am not sure what is right for your system. This serum then protects cell integrity and also helps them to attach. The process is a bit of trial and error. Occasionally one can use a pipette to detach the last 15% of attached cells but I probably would not worry about these cells as then the rest of the detached cells may be affected. http://www.rndsystems.com/literature_ccm004.aspx Cheers

Kudos to guys for valuable information, and detailed answers. I do agree the complexity of this kind of quantitative data generation. I went through many bioinformatics tool to predict this kind of data sets.I aware that,a basic signal cascade depends on multi factors of a system, and also science said specificity gains rank.In that view we predict this, X- kind of agonist molecule undergoes in Y -kind of signalling results Z as a result, on contrary also explains (involvement of xyz and x', y',z') differential mechanisms during individual cell type also. The complexity known to us, a single type of cell was guided by many kinds of signals at randomly back to back by many signal gateways, as a reference the complexity re-farms and maintains up regulations or down regulations. I am happy for "Thomas Lettner" comment and for very detailed view with his mind, and Maria Thomas, cheers. For the moment my topics were designed to study Co infection immune response in a defensing cell type In vitro. But this question in my mind developed many points to resolve before the effect conclusion. Perhaps, I can try an individual data set for each condition, chart of prediction.as a secondary i mix them all to see which gains high values(Rank). Gunisha, thanks for the wonderful tool, and Robert ..cheers Currently reading, as much of Ravi Iyengars laboratory articles, and also enrolled an online course with him in coursera. Perhaps,i will focus little time to generate this kind of pathways with my computer. Cheers and Thanks, Sriki

After FACS cell sorting for CD34+, collect the cells in sterile tube and grow with DMEM F12(1:1) and B27 media (Invitrogen). Try.

You can isolate the CD133+ cells with magnetic cell sorting method (MACS), (Check Miltenyi Biotec, methodology) and grow again with media. Then collect these cells and check for % CD133+ cells by FACS cell sorting. Then collect in sterile tube to grow again with suitable media. It is better to do for two or three CD+ markers instead of one CD marker for any specific type of cell of interest.

Cheaper than this: “Isolation of nuclear fractionation from Parasites” This protocol is applied to low amount of organisms…might work Parasites were lysed in a hypotonic cytoplasmic lysis buffer CLB (20mM HEPES (pH7.9), 10mM KCl, 1mM EDTA, 1mM EGTA, 0.65% NP-40, 1mM DTT, Complete TM protease inhibitors (Roche Diagnostics)) for 5 min on ice. Nuclei were pelleted at 2,000g for 5 min and the supernatant saved (cytoplasmic extract; fraction 1). After four to seven washes in CLB nuclei were extracted in 1.5ml (ring stages and trophozoites) or 3ml (schizonts) low salt buffer LSB (20mM HEPES (pH7.9), 0.1M KCl, 1mM EDTA, 1mM EGTA, 1mM DTT, protease inhibitors) for 20 min at 4°C under constant agitation. Insoluble material was pelleted for 3 min at 13,000rpm and the soluble fraction saved (low salt extract; fraction 2). Pellets were washed twice in 1.5ml (3ml) LSB and chromatin was solubilised in 1.5ml (3ml) DNAseI-digestion buffer DDB (20mM Tris-HCl (pH7.5); 15mM NaCl, 60mM KCl, 1mM CaCl2, 5mM MgCl2, 5mM MnCl2, 300mM sucrose, 0.4% NP-40, 1mM DTT, protease inhibitors) containing 100-500U DNAseI for 20 min under constant agitation at RT and 37°C. Samples were centrifuged at 13,000rpm for 3 min and the soluble fraction was saved (DNAseI extract; fraction 3). After two washes in 1.5ml (3ml) DDB pellets were extracted with high salt buffer HSB (20mM HEPES (pH7.9), 1M KCl, 1mM EDTA, 1mM EGTA, 1mM DTT, protease inhibitors) for 20 min at 4°C under constant agitation. The soluble fraction was recovered by centrifugation at 13,000rpm for 3 min and saved (high salt extract; fraction 4). The insoluble nuclear matrix fraction was washed twice in 1.5ml (3ml) HSB, solubilised in 1.5ml (3ml) SDS extraction buffer SEB (2%SDS, 10mM Tris-HCl (pH 7.5)) for 20 min under constant agitation at room temperature, cleared by centrifugation at 13,000rpm for 15min and saved (SDS extract; fraction 5).

Hi, Maybe FACS and then cell sorting may help. You could check these links: http://www.advancedcell.com/documents/0000/0238/rpe_mie.pdf http://www.utoronto.ca/nbrg/pubs1/2012_Clarke_Euro%20Neruo.pdf Hope it helps!

hi DMSO is best solvent but remember these points also while solubilize start checking your best solvent with mixture of two solvent system for i e, 1%, 5 %, 10%,25 %,50% like that you gradually increase the solvent mixture while solubilize herbal extract for minimize the effect of DMSO on the test

I usually prefer not to use wells as I have the same problem, much better T-25 flasks, but I had good results with low-adhesion wells from Corning. I think that it's also a question of area/concentration, the last one in my experience, should not to be higher than 500.000 cells/mL and not lower than 250.000, anyway i would use lower concentration if I want to work in a 6 well. I totally agree with Tamara and, if I can made a consideration, neurospheres can drive you crazy at the beginning but with a bit of practice it will be ok.

You can use Griess reagent assay which is based on colorimetric approach.

Dear Jose, I think the challenges of iPS in the next 10 years would be dealing with the epigenetic memory of the reprogrammed somatic cells that might limit their potential for therapeutic use, or briefly to optimize the reprogramming process.

What is exactly the Tm of your primers? And which algorithm you use to determine the Tm? Each software use different algorithms, so you estimate a Tm which is much more different than the real. I use Gradient PCR from 50-70 when I have a Tm around 58. With different concentrations of MgCl2 (1,5-2,5).

Csn you sort the cells? If they are CD133 PBMCs, they could probably be cultured on fibronectin coated plates + EBM. See article by Suuronen, 2006. http://cardiovascres.oxfordjournals.org/content/70/1/126.full.pdf+html

Hello, first things first! you need to have your controls. 1. if you want to determine whether the molecule A, directly or indirectly affects the expression of the molecule B, than you need to have a control of the same cell line, cultured in the exact same way and transfected with a negative control that is not supposed to alter the expression of molecule B. Only then can you tell that mol A affects mol B (by quantifying the expression of you protein B with qPCR, western blot or luciferase). 2. Now if you have established that mol A affects the expression of mol B, and you're looking for a direct interaction between both proteins, you can do colocalization experiments or yeast two-hybrids (both a way more time consuming than qPCR or western). Hope it helps!

Please let me know if you need more information.....wish you Good luck !

How many cells in total or per µl do you need for your experiments? Then you can calculate how much peripheral blood you should start with. Generally, 3 ml Ficoll-Hypaque per 10 ml blood/PBS mixture are used. You may also want to have a look at the information here: https://www.researchgate.net/post/Hello_Pleas_I_need_to_isolate_the_peripheral_blood_mono-nuclear_cells_called_buffy_coat_PBMC_MNC_from_blood_using_Ficoll_Is_there_any_protocol

Thanks for the protocol. That was very helpful.

Hai guys, what cell lines is the best for insulin secretion study.

Yes. Very difficult. Requires extensive cell biology and cell biophysics knowledge, the same of gradient chemistry, a very pure population or one in which the stem cell of interest is dramatically different in size, lipid content, cell mass, and many other variables.

The best way to answer your question to create a basic model of an ESC, and then look for parallels. ESCs by definition: Are derived from a blastocyst. Require a feeder bed to survive, much less expand. Said feed bed is most effective if foreskin fibroblasts, and the cytokines they produce. Requires serum and other factors. Are immortal, but subject to phenotypic stem cell degradation with passages, genomic instability, etc. Are fully potent, in a trigerm layer manner, to create any cell of any tissue that at least has a semblance of said cell in its true biologic phenotype. Express key TFs (5 - 7), required for its maintenance of immaturity, prevention of differentiation, etc. So called NOS transcription factors - Nanog, OCT3/4A, SOX2, and others such as Stat2, and C-myc, etc., which regulate huge diverse and overlapping gene protein cascade biologics intracellularly for stem cell biology or maturation and organization with purpose. Are responsive in species specific manner to growth factors (i.e., LIF, murine) Are best modeled by taking an ESC population, influencing it towards a Neural Stem and Progenitor and Terminal Lineage phenotype of the Nervous System in vitro. Well characterized, published. There are adult stem cells, cord stem cells (likely 7-8 different stem cell populations), amniotic, bone marrow, adipose, MSC, and on and on. Adult Stem Cells can be regressed through stringent stem cell biologics and manipulation, and artificially as well. In my opinion, there is little strong evidence of proof of concept for ESCs and the second most common studied stem cell, the MSCs. Not beyond the artificial in vitro model that use molecular and cell biology, not actual tissue or organ biology, to suggest unlimited potential with limited success. That being said, any stem cell population that can, at the gene and protein level, be shown to have expression profiles that are close to that of a normal healthy ESC, is the cell you are looking for. Interestingly, there are reports of ESCs becoming one step committed, and adult stem cells being regressed to and ESC phenotype, and the common denominator is CD133. The neural stem cell induction model with ESCs suggests that an ESC high in its expected gene expression profile, but low in CD133, is the true early ESC, but when CD133 is upregulated, it has taken one committed step but is still for all intensive purposes of an ESC phenotype. Several publications. The question is, whether the reverse is true for a CD133 population of adult stem cells induced, coerced to coexpress ESC NOS TFs and related proteins. Have you created from a single germ stem cell of adult origin, an early ESC progenitor, multi germ potential, cross germ therapeutic potential, or even more simple, same germ same tissue same mature tissue lineage potential, for stem cell therapeutics and tissue engineering.

This article may help. They describe their basic protocol for isolation and culturing of human adipocytes. http://diabetes.diabetesjournals.org/content/58/7/1550.full.pdf+html Also, our department frequently isolates adipocytes for energy balance studies. I will dig around to see if I can find a protocol.

There could be several reasons for the center necrotic colonies. As many of them mentioned it could be due to the hypoxic environment prevailing in the center. Apart from that there could be several other reasons like poor feeder quality, inappropriate feeder density and/or low LIF concentration, use of expired media, etc.. Generally large colonies with necrotic centers also happens to cells which were not passaged for several days.

It would be ideal if you can review this recent paper from nature communications: http://www.nature.com/ncomms/journal/v3/n6/full/ncomms1867.html?WT.ec_id=NCOMMS-20120606 They used good techniques to validate their findings. Although it originated some controvery in the vascular field: http://circres.ahajournals.org/content/112/1/17

Dear Marlene I was not commenting your answer but replying to Hadi's question.

The question about circulating-migrating dental pulp stem cells during repair (but also during homeostasis...) is interesting and important. A definitive answer could be given only after carefully designed experiments. Anything else is speculation, based on scientific work on other tissues/organs. It is well known that teeth are not liver, heart, bones etc... Everything is different in the body, specialized stem cells and niches can be detected in specific tissues/organs.

I have heard that high ratios of FBS/FCS will effect MSCs and will reduce their stemness characteristics. In other words, MSCs might undergo differentiation and with that being said, I'll go with 10 to approximately 15% of serum. However, as FBS/FCS might induce apotheosis to cells, ITS is a great substitute for cultures.

If you are talking about freshly isolated UC-MSCs I have a very similar experience that they do not express much CD105 upon digestion of the Wharton's Jelly. They should upregulate CD105 to over 90% by P3 or P4. That said, every now and then you will have a donor which doesn't upregulate a particular molecule. This might be your situation, but over n=4 CD105 should be higher.

Could you also grow your cells on slides or coverslips placed in a Petri dish? In either case, for hypotonic treatment and fixation you have to carefully soak off the medium and slowly add hypotonic solution; after reincubation at 37°C again carefully soak off the hypotonic solution and add fixative (methanol/acetic acid). It might be best to directly add some fixative to the hypotonic solution, replacing it step by step with cold fixative, otherwise your cells might detach from the surface. Fix the cells in the fixative in the Petri dish at -20°C, then remove the fixative and air dry the cells. Then I would employ a standard FISH protocol; however, I am not familiar with Cambio probes. I would select the area you are interested in and just add the FISH solutions to this area and use coverslips as you would do for a conventional FISH on slides. I think you have to make sure that your Petri dishes have no autofluorescence, may be you have to use a brand suitable for life imaging, may be glass bottom dishes, however, I am not sure whether this will indeed be necessary.

Thanks Wanda (Miles), glad to be of help, and thanks also to Guruprasad Gopalakrishnan and Joachim Pimiskern for your comments. You are correct that kynurenine and the kynurenine pathway (KP) exhibit "Janus-like" or dual modes of activity depending on the context, with a positive role in: infection control, pregnancy, transplantation acceptance, autoimmunity (especially in non-obese diabetes and systemic lupus erythematosus (SLE)), and allergy control among others, and a decidedly negative role in: malignancies (especially endometrial, colorectal, lung, esophageal squamaous cell and vulvar squamous cell carcinoma’s , and in virtually all brain tumors, among other cancers). In addition, kynurenine pathway dysregulation plays a significant role in non-malignancies such as Alzheimer’s disease, schizophrenia, depression, Huntington and Parkinson disease, and age-related cataracts. SOME CAUTION Trials therefore must exert caution to avoid potential secondary / reflexive adverse effects of kynurenine pathway (KP), and IDO / TDO, inhibition, part of which can be accomplished by deployment of highly selective rather than broad-spectrum inhibitors. To date however it should be noted clinical trials have not yet uncovered significant adverse events although we should continue to avoid what I would designate at risk populations such as SLE patients, pregnant women, or any patient with active or at-risk infection, until mature data provide highly targeted selective inhibition strategies. KYNURENINE PATHWAY (KP) INHIBITION IN CLINICAL TRIALS Now, as to therapeutic goals, let me sketch these out within primarily the oncology context, although progress is emerging in non-malignancy disorders too: In dealing with kynurenine pathway (KP) research, I divide cancers into two large groups depending on whether they are driven by the IDO versus the TDO component of the kynurenine pathway (right now, the third component I noted above, namely ACMSD, is no as yet being pursued fully). Remember that IDO and TDO respond to different activating stimuli, with IDO but not TDO the expression of IDO - but not TDO - being upregulated following pro-inflammatory cytokine challenge. So the groups are either: (1) the class of IDO-inhibitor targeting cancers such as Non Small-cell Lung Cancer (NSCLC) and Chronic Lymphocytic Leukemia (CLL), or (2) the class of TDO-inhibitor targeting cancers such as malignant gloimas. But there are I should note cancers that are both IDO- and TDO-driven such as Head and Neck Cancers (HNC) in which we would speculate that concurrently inhibition of both IDO and TDO might prove of benefit. There is more research activity in the IDO-inhibitor targeting class: it's known that IDO directly suppresses the effector T cell proliferation and differentiation, while also markedly enhances the suppressor activity of regulatory T cells (Tregs), suggesting that IDO-inhibitor drugs can both: (1) enhance anti-tumor immune responses making them attractive for anti-tumor vaccines and other immunotherapy, and (2) they are also synergistic with many chemotherapeutic drugs, so that chemotherapeutic synergy is another exploitable strategy. An example of the first IDO inhibitor strategy, namely of enhancing anti-tumor immune response, is the IDOvaccine Trial (NCT01219348) recruiting in Denmark. An example of the second IDO inhibitor strategy is the IDO MBC Trial (NCT01792050) which is exploring the potential synergy of the IDO inhibitor Indoximod when used in combination with docetaxel (Taxotere) chemotherapy for the treatment of metastatic breast cancer (MBC); the trial is in-progress at the H. Lee Moffitt Cancer Center and Research Institute in Florida. One intriguing trial is the IDO/Survivin Peptide Vaccine Trial (NCT01543464) which combines both approaches - IDO anti-tumor immune response enhancement and chemotherapeutic synergy - by combining the IDO/Survivin Peptide Vaccine with temozolomide (TMZ) in treating metastatic malignant melanoma, now recruiting in Denmark. And the INCB024360 EOC Trial (NCT01685255) takes the direct antitumor approach, comparing the IDO inhibitor INCB024360 (from Incyte Corporation) to tamoxifen in epithelial ovarian cancer (EOC), and is now recruiting in multiple U.S. locations and in Canada. In addition, 1-D-MT Inhibitor Trial (NCT00567931) uses 1-methyl-D-tryptophan (1-D-MT, also called D-1MT) which is an IDO inhibitor, in the treatment of patients with non-resectable metastatic or refractory solid tumors; now recruiting in Florida and Virginia. OTHER APPLICATIONS, AND THE EMERGENCE OF NATURAL KP INHIBITORS As to TDO (tryptophan 2,3-dioxygenase) inhibitors, the Belgium team of Luc Pilotte and colleagues (Proc Natl Acad Sci U S A. 2012) have completed a proof-of-concept preclinical study of the reversal of tumoral immune resistance via a TDO inhibitor. My own research in integrative oncology find the green tea component EGCG behaves as a natural IDO inhibitor and this has been confirmed by Kengo Ogawa and colleagues in Japan [Oncol Letters 2012] where EGCG may exert antitumor effects on colorectal cancer (CRC) in part by inhibition of IDO expression and function via the suppression of STAT1 activation, adding more evidence that EGCG may serve as a potential natural antitumor immunotherapy agent for the chemoprevention and treatment of CRC, findings that are motivated by the fact that high IDO expression levels of IDO are associated with disease progression and poor overall survival in CRC patients, as demonstrated by Liesbeth Ferdinande and colleagues in Belgium (Br J Cancer 2012). In addition, Sophie Chen and colleagues in the UK [Biochem Biophys Res Commun. 2012] have demonstrated that flavone phytochemicals apigenin, baicalein, chrysin, and wogonin can also provide anti-cancer and neuro-protective activity via IDO inhibition, thus also serving as natural IDO inhibitors. And Helen Ball's team at the University of Sydney [Bakmiwewa et al., Bioorg Med Chem Lett. 2012] has recently that certain proton pump inhibitors (PPIs) can serve as selective IDO inhibitors, as can certain COX-2 inhibitors. What this suggests is that certain selective and demonstrably safe natural KP/IDO/TDO inhibitors can be cautiously exploited until clinical trials complete and commercially viable agents enter the marketplace (highly standardized EGCG for example, or a combination of EGCG + celecoxib serving as a dual-IDO selective inhibitor complex that may prove of benefit in advanced cancers or otherwise unsalvageable malignant stages where research has already confirmed the absence of appreciable harm). And within the next 18 months we will experience something of an explosion of research and clinical trials further refining our understanding of the complexities and nuances of kynurenine pathway in cancers and in non-malignant disorders. Constantine Kaniklidis Director of Medical Research, No Surrender Breast Cancer Foundation (NSBCF) European Association for Cancer Research (EACR)