I have generated the iPSC using SeV [imaged attached]. The iPSC is positive for Tra-1-81. I picked the cells manually for further expansion but my cells neither attached nor expand. I have gone through several articles but confused with partially and fully reprogrammed iPSCs. Each paper has their own explanation. Few say, the image I attached is partial and other say it is fully reprogrammed.
It will be great if an expert in the field could explain the morphology of iPSC.
Do the fibroblast from an old age [70yrs] skin biopsy affect the reprogramming?
Can anyone help with suitable protocol for reprogramming, picking, and initial expansion?
Thank you.
Love&Hugs
Rupendra Shrestha
Hello, I have attached a protocol from Nature Protocols that was very helpful to me when I was generating iPSC from fibroblast biopsy. Their troubleshooting table on page 21 suggests that the initial colony you are plating may be too big which hinders attachment, and they recommend cutting the colony into smaller pieces before harvest so each piece has more surface area to attach and expand from. Hope this is helpful, best wishes for your experiments.
Thanks a lot, Pollan S.G. for sharing the ideas and relevant article. I will follow the protocol explained in the article. I tried colony plating like small cut pieces, big colony and single cell suspension too.
Thank you again
Hello, looks like that your colonies grew up to much (lost passage time) and/or your MEF concentration is to high (no space to your colony iPS grow). You can try make passage (fragment colony) when colony has size of 100x (in microscopy) and/or use MEF at 50 to 100 k/one well of 12 plate. If you can, use geltrex or matrigel with E8 or mTeSR1 after reprogramming in MEF and ESC homemade medium. Use NaB - Sodium Butyrate in medium until 5th passage will helps your reprogramming (works like VPA). We do this in our lab. Good lucky.
We follow these articles:
Generation of integration-free human induced pluripotent stem cells from postnatal blood mononuclear cells by plasmid vector expression.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3756815/
A Facile Method to Establish Human Induced Pluripotent Stem Cells From Adult Blood Cells Under Feeder-Free and Xeno-Free Culture Conditions: A Clinically Compliant Approach
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4367508/
Efficient human iPS cell derivation by a non-integrating plasmid from blood cells with unique epigenetic and gene expression signatures
http://www.nature.com/cr/journal/v21/n3/full/cr201112a.html
Hi Rupendra,
I would say your colonies seem to be differentiating in the middle (the dense parts) and soon will die. Otherwise the colonies seems to have clear borders, which is most of the time a good sign. The partial reprogrammed cells tend to have a different morphology, and not necessarily grow in a colony. Nevertheless I culture my iPSC on matrigel, so feeder free, and had done the SeV reprogramming the same way. Therefore I cannot comment much further on the on-feeder cultures.
Another point would be to cut them relatively smaller but definitely not single cells, as even with ROCK inhibitor single cells mostly cannot survive. Having smaller fragments at the time of plating and passaging on time might improve your culture.
Good luck,
Ceren
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.
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 )"
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
Good luck,
Justyna
Chapter Induced Pluripotent Stem Cells (iPSCs): An Emerging Model Sy...
Thanks lot to everyone for their input. I am sharing the image of feeder-free iPSC with high efficiency rate.
That photo still shows a lot of differentiation to clean up over the next couple of passages. If you continue it without doing so the ratio of differentiated cells will increase.
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