Dear Chandra Kishore,

iPS cells can be established from a variety of fibroblast cell lines deposited in cell banks. I have established iPS cells from a fibroblast cell line called HFL1. It is not necessary to collect fibroblasts from individuals.

It has been reported that some cancer cell lines can also be initialized.

(Of course, they are not normal pluripotent stem cells.)

However, since some cultured cell lines have high efficiency in establishing iPS cells and others have low efficiency, it is assumed that it is better to proceed with initialization experiments using multiple cultured cell lines or to use fibroblast cell lines that have been reported in papers.

Best

Narumi

Thank you Dr. Narumi Uno. I have another follow up question. For the generation of organoid, can we use human dermal fibroblast reprogrammed iPSC cells or do I need iPSC cell line?

I am sorry, but I do not understand some of your intentions.

What is the difference between human dermal fibroblast reprogrammed iPSC cells and do I need iPSC cell line?

Do you mean iPSC cells that you have generated yourself or cell lines that can be purchased from cell banks or vendors?

Any iPS cell line that has been shown to be undifferentiated by expression of undifferentiated markers or assessment of teratoma formation can logically be considered to be capable of organoid formation.

But unless there is a special reason, it would be better to use iPS cell lines that have been used in similar papers and are available from cell banks.

This special reason means that you have iPS cells derived from a disease patient with a specific genetic mutation.

In other words, there is an experimental design issue.

Due to epigenetic memory and other factors, each established clone of iPS cells has a different gene expression level and differentiation potential. Even if undifferentiated state can be confirmed by the above evaluation, detailed molecular dynamics and other factors tend to differ. The efficiency of organoid formation is also likely to vary greatly from clone to clone. Therefore, it is presumed that using iPS cell lines whose organoid formation ability has already been confirmed in the previous report would be a lower hurdle in the experimental design.

Clonal variations in each iPS cell line and, moreover, differences in genetic backgrounds such as SNPs due to the different individuals from whom the lines were established and the normal cell lines to be compared may create complications in data interpretation.

Best,

Narumi

Dear sir, iPSC cells, I mean the iPSC cells that we will generate in our lab from human dermal fibroblast cells. iPSC cell line, I mean commercially available iPSC cells that can be contiously propagated.

Thank you for your explanation.

If that is the case, then it is as I interpreted it earlier, so I don't see any problem with the rest of the text in my earlier question.

Thank you sir for your clear explanation.

Sir on the same line, how important is passage number of primary human dermal fibroblast or fibroblast cell line for efficiency in iPSC generation and passage number of iPSC cells/iPSC cell line for organoid generation efficiency?

When iPS cells are cultured for a long period, chromosomal aberrations may occur in the iPS cells. In addition, clones with chromosomal abnormalities may appear during reprogramming.

Therefore, when iPS cell clones are obtained by reporogramming experiments, it is desirable to evaluate that they have a normal karyotype by CGH and Karyotyping.

The iPS cell lines that are available for sale have probably been evaluated for karyotyping normality.

Other factors may include changes in methylome through passage culture. The reagents and equipment used in the culture will have a significant affect on the differentiation potential of the iPS cells. Furthermore, even who cultured the cells may be a factor in the methylome changes.

There are two main methods for establishing and culturing iPS cells: using feeder cells and not using feeder cells. A variety of iPS cell culture media have been reported and marketed, but the choice of which method to use should be fixed. In addition, it should be investigated whether organoid differentiation potential has been reported when iPS cells are passively cultured using this culture method.

As described above, passage culture can change the properties of iPS cells, but not immediately after several passages. On the other hand, one should be cautious about comparing values obtained from experiments conducted by others, since who conducted the experiment may have a significant affect on the results.

By the way, I will send you an iPS cell establishment method distributed by my previous collaborator.

(This is an advertisement.)

Best,

Narumi

Thank you sir

agre with Jaba above - if a cell is 'immortalised' to become a cell line, its genome is typically altered, some-what cancerous. not a good place to start with generating an iPSC cell line from. If you do use a line to make an iPSC, i would advise you get the resulting iPSC line checked out through a Next Gen sequencing approach or similar technique.