Hi Daniel,

Which reprogramming system did you use? I guess you already manually picked your colonies and are trying to expand your single "clones"? If so, did you check your colonies for pluripotency? If yes, did you check for pluripotency again after several passages? What is your split ratio/seeding density? What I see in my cells (human dermal fibroblast derived iPSCs) is an increased spontaneous differentiation when the seeding density is too low. Did you check for Mycoplasma contamination? 

16 Days in one passage sounds like a very long time to me. My cells need a split every 5-7 days (depending on cell line and split ratio).

Best regards

Thomas

Hi,

Try to use the Alkaline Phosphatase Live Stain for screening colonies during early stages of the reprogramming. Alkaline Phosphatase stain iPSC colony with good phenotype.  

Choose stained with the alkaline phosphatase one colony. Perform clonal selection.

You must to obtain hiPS cell line from one single colony with good phenotype. After propagation check main pluripotent marker at protein and mRNA level.

Good luck!

Thank you all for your quick replies!

Thomas, 

I have used a lentivirus transduction with OCT4, SOX2, NANOG, Lin28, KLF4 and C-MYC. I used AP live staining to check pluripotency but the signal was too weak or doesn't appeared at all. I picked manually colonies with better shape (not well defined edges) and seeded them in 75000 cells/cm2 of SNL feeder layer. I have used this feeder cells since it were previously used in this lab, but I not sure if MEF would be better. Has anyone any experience with this feeder layer?. Also the bFGF conc. seems to be a bit lower than other protocols suggest...   

Hi,

In my opinion PBMCs  cells were not completely  reprogrammed. 

I added pictures from: M. Diana Neely, Andrew M. Tidball, Asad A. Aboud, Kevin C. Ess, Aaron B. Bowman, Induced Pluripotent Stem Cells (iPSCs): An Emerging Model System for the Study of Human Neurotoxicology, Cell Culture Techniques, Volume 56 of the series Neuromethods pp 27-61

https://www.researchgate.net/publication/225844841_Induced_Pluripotent_Stem_Cells_iPSCs_An_Emerging_Model_System_for_the_Study_of_Human_Neurotoxicology

Maja Diana Neely shown (Fig.4.): "examples of various colony morphology types observed during reprogramming and propagation of iPSC colonies on feeders and Matrigel. ( a–c ) Partially reprogrammed human iPSC colonies form circular associations of cells appearing either phase bright ( a ) or phase dark ( b , c ). They usually do not show clearly defined colony margins and show an overall fuzzy appearance. The morphologies of these colonies are in sharp contrast to the smooth colony margins and flatter appearance of true iPSC colonies. ( d , e ) Human iPSCs derived from the fibroblasts of a normal subject are growing on feeder cells. These images display one human iPSC colony with surrounding SNL feeder cells. Human iPSC colonies can have a circular or oval/tear-drop shape. ( f )" 

Chapter Induced Pluripotent Stem Cells (iPSCs): An Emerging Model Sy...

I added pictures from: M. Diana Neely, Andrew M. Tidball, Asad A. Aboud, Kevin C. Ess, Aaron B. Bowman, Induced Pluripotent Stem Cells (iPSCs): An Emerging Model System for the Study of Human Neurotoxicology, Cell Culture Techniques, Volume 56 of the series Neuromethods pp 27-61

https://www.researchgate.net/publication/225844841_Induced_Pluripotent_Stem_Cells_iPSCs_An_Emerging_Model_System_for_the_Study_of_Human_Neurotoxicology

How to improve quality of cultured IPSc from PBMCs? - ResearchGate. Available from: https://www.researchgate.net/post/How_to_improve_quality_of_cultured_IPSc_from_PBMCs#589dbe0ceeae39453c734e68 [accessed Feb 10, 2017].

Chapter Induced Pluripotent Stem Cells (iPSCs): An Emerging Model Sy...

Hi Daniel,

I'm afraid your reprogramming didn't work as it should have. The weak/non existing AP live staining is a bad sign. If I were you I would make an additional staining with Oct4/TRA-1-60/SSEA4 with your clones. If all 3 are expressed, than there is a chance to save your cells, if not than you most likely don't have iPSCs... But the morphology indicates that they won't have those markers.

We used mRNA based reprogramming for our fibroblasts (Miltenyi Biotec; no affiliation) and it worked like a charm (also the first time I did this). I can't say anything about the bFGF concentration in the iPS medium, because we buy it "ready-to-use" and the concentration is nowhere to be found (MACS iPS Brew, XF).

I tested cultivation on MEF Feeders and hESC Matrigel ECM and there was no big difference, regarding undifferentiated status, proliferation or morphology, so we are using Matrigel for our cultivation, because of its advantages.

I attached a Picture with two of our colonies on the "Primary plate", before picking on MEF Feeders. Scale bar: 200 µm

Best regards and good luck

Thank you so much Justyna!

Very nice paper. Certainly, our colonies looks like not completely reprogrammed. I will carefully check the reprogramming part of the protocol. 

Thank you all, your answers were really useful. 

Best,

Daniel

Dear Thomas Klein,

While I work with naturally occurring adult-derived pluripotent stem cells (as well as totipotent stem cells, germ layer lineage stem cells, and progenitor cells), rather than induced pluripotent stem cells, the basic principles are pretty much the same. True stem cells (totipotent stem cells, pluripotent stem cells, and germ layer lineage stem cells) have three activity states: quiescence, differentiation, and proliferation (in that order). And these states do not overlap.  

Pluripotent stem cells (at least the ones I work with) will not spontaneously differentiate in culture in the absence of inductive factors. Their default state is quiescence, unless undergoing proliferation. The spontaneous differentiation program you are seeing is probably driven by unrecognized inductive factors contaminating your culture medium. These factors are usually within the serum, unless you are using recombinant factors in your medium such as TGF-b or b-FGF to induce proliferation. Be extremely careful using growth factors, except for a very few of them (i.e., PDGF-BB which will only stimulates cellular proliferation), most have multiple activities dependent on whether you are culturing progenitor cells or stem cells. For example, other research groups use TGF-b and b-FGF to stimulate proliferation in their mesenchymal stem cells. The cell type identified as a mesenchymal “stem” cell is actually a tripotent progenitor cell that is already committed to forming three tissue lineages: cartilage, fat, and bone. Since the progenitor cell has already committed to specific tissue types it will not respond to any inductive factors inducing differentiation into other tissue types.  Therefore it will enter the last functional state, which is proliferation. True stem cells, on the other hand such as pluripotent stem cells, will respond to inductive factors across ectodermal, mesodermal, and endodermal germ layer lineages. For example, pluripotent stem cells will form fibroblasts in the presence of TGF-b or b-FGF, endothelial cells in the presence of VEGF or a-FGF, red blood cells in the presence of EPO, cartilage and bone in the presence of BMP-2, hepatocytes in the presence of HGF, and neuronal cells in the presence of NGF.    

If you use a serum that is not heat inactivated or serum-free defined (without inductive factors) it probably contains inductive factor(s) that will cause induced lineage-commitment in lineage-uncommitted pluripotent stem cells. The induced phenotype(s) usually do(es) not show up until after you passage your cells. You can force the issue of induced phenotypic expression by treating your cultures with serum containing insulin (2 micrograms/ml in addition to what is in your medium). In this respect you will see phenotypic change within 1-4 weeks in culture and usually before passaging.

Before we changed to a specially prepared heat inactivated serum my lab screened over 300 lots of serum looking for absence of inductive activities. Within those 300+ lots of serum we only found 4 lots that were absent of factors causing our pluripotent (totipotent, and germ layer lineage) stem cells to “spontaneously” differentiate in culture. Once identified, we proceeded to buy the entire lot of serum, froze and stored at -80C. I am sure you are wondering why freezing and storage at -80C and not at the manufacturer’s recommended  storage temperatures od +4C or -20C. Freezing at the lower (-80C) temperature puts the serum into a suspended animation state in which it will not deteriorate. At higher temperatures (+4C and to a lesser extent -20C) the serum will begin to degrade (expiration date). Our normal procedure was to buy the entire lot, aliquot the amount of serum we would need for a standard experiment, freeze and store at -80C, and then only remove from storage the amount of serum we would need for the experiment. Repeated freeze/thaws will accelerate degradation of the serum. But above all, do NOT use a frost-free freezer to store any material containing proteins (serum, recombinants, antibodies, etc.). To maintain the frost-free state the freezer will repeatedly undergo multiple thaw/freeze cycles to reduce frost build-up. In this respect we could standardize our experiments to the same lot of serum and not worry about variability due degradation or contaminating material.

Our method of testing the sera was to use clones (derived from single cells) of pluripotent stem cells and totipotent stem cells with serum at 1%, 3%, 5%, 10%, 15%, and 20% v/v with our medium, including known positive and negative controls. Included in the medium was 2 micrograms insulin/ml. (Insulin is a progression factor that accelerates phenotypic expression in induced lineage-committed cells. So if there are any inductive factors present in the serum you will see a phenotypic change in your cultures before passage.) To verify change in phenotype of the cells we used our ELICA assay. This assay allows one to quantify, visualize, and standardize to DNA content (nanograms phenotypic expression marker per microgram DNA) within the same well of 96-well plates. Using 96-well plates we could screen 12 lots of serum simultaneously.

One needs to know their model system inside and out before being comfortable with experimental results. Everything we discovered concerning our adult-derived stem cells and progenitor cells was determined empirically. The above are just a few friendly suggestions to circumvent potential problems you will probably encounter.  All my publications are in pdfs and posted on this website for public download. I attached our methods paper for the ELICA assay. Good luck with your studies. If you have any additional questions, just ask.

Dear Henry,

Thanks for your interesting comment.

We are using a feeder/serum free iPSC system. iPSCs cultivated on hESC qualified Matrigel (Corning), cultivated in MACS Brew iPS XF (Miltenyi Biotec), with the addition of Y-27632 (ROCKi) for the first 24 hours after splitting. Spontaneous differentiation of iPSCs unfortunately is a common and annoying thing to happen, especially when you let your cells grow confluent, or don't change your medium daily. So my guess would be (I am by far no expert in this field and I am sure it is not that easy) that the accumulation of metabolistic waste products in the medium can induce certain signaling pathways, leading to this spontaneous differentiation. Fortunately there is a small time frame in which you can "save" your iPSCs (when you have an important line and only few backups in the nitrogen tank, e.g. during clonal expansion), but usually when I see too much differentiated cells at the colony borders I throw this line away and get another aliquot from the N2 tank.

There are also commercial solutions, like "ReLesR" (Stemcell technologies) to seperate your undifferentiated iPSCs from party differentiated cells on your plate. We gave it a try but it didn't really work for us (could be, because it is optimized for use in mTesR medium, which we don't have).

I personally don't add any growth factors/serum to my iPSC culture for routine maintenance. As said we are purchasing the medium from a company, add the provided supplement and use it as it is.  We have to trust the company and their quality assurance. Screening of different lots and then buy a huge amount of a specific lot is not suitable, because of the short shelf-life of the medium (and the financial aspect). I don't have the numbers for the pure medium/supplement here, but supplemented medium for example is stable for 2 weeks @4°C or 2 months @-20°C and you can really see that your cells behave differently with older medium.

For (neuronal) differentiation studies we are relying on small molecules to induce the differentiation and only add recombinant growth factors for the maturation of the differentiated neurons.

The only time my cells come in contact with serum-like reagents (e.g. KnochKout Serum Replacement) is for freezing (10% DMSO in KO Serum Replacement), or for 1d during the conversion of iPSCs to Neural Progenitors and I am very happy that I don't have to use FCS, since I heard a lot of horror stories about "bad lots".

But an interesting read!

Dear Thomas,

Have you tried conditioned medium from undifferentiated cultured cells? Keep your spent medium and add fresh medium at ratio of 1:1. At next media change use mix of spent-fresh. Repeat procedure to get 3-4x conditioned medium. The exosomes in conditioned medium by that time are at sufficient quantity to rescue sick cells.

If you are still having problems with spontaneous differentiation, you could add LIF, leukemia inhibitory factor, to your media. But beware, LIF is cell number-specific, you need to titrate amount based on cell numbers to keep the cells from spontaneously differentiating.