Ex-Vivo Cell Tracking

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Ex-Vivo Cell Tracking

Postby GabeA23 » Jul 26 2012 7:08 pm

[This post is from 2012. It has been revamped in 2014. Click here to go to the first of the new posts.]

Hello fellow scientists!!

I am the lead researcher of a group that is trying to produce a functioning tissue-engineered ligament for anterior cruciate ligament regeneration.
Anyway... we are currently using a rat model and implanting "scaffolds" (basically a polymer construct) with cells seeded onto them into the knee capsule. I am trying to figure out how to "track" the cells to evaluate survival and proliferation after we sacrifice the animals. By "tracking" I mean differentiate between our implanted cells and the host cells that infiltrate our construct. There are two transplantation models I am trying to figure out:

1) Rat cells into rats. We have GFP positive fibroblast cells (harvested from a transgenic GFP rat) that we implant into normal (non-GFP) animals (for weeks to months), but there are a whole host of problems trying to actually TRACK these GFP+ cells in post-mortem histological sections. The biggest problem is that after a few passages in culture, these fibroblasts hardly transcribe the GFP gene anymore. If I take a flask of these GFP+ fibroblasts and put them under a fluorescent scope, I don't see any green fluorescence. So tracking the cells in-vivo using their endogenous fluorescence is a no-go. Next we took tissue-sections of our implants and tried anti-GFP immunohistochemistry (IHC) using a primary antibody against the GFP protein and a fluorescent secondary and once again saw basically no GFP signal (probably due to extremely low levels of GFP expression by the implanted cells). Now, we know we have cells in our constructs because we can do PCR and detect the GFP gene, but we want to visualize the distribution of cells.

Any ideas for alternative cell-tracking techniques (with or without relying on GFP) would be greatly appreciated. Do any of you have any experience with FISH? Could I use FISH to visualize the GFP genes in our construct? What about CM-Dil? I'v heard mixed reviews about CM-Dil and heard that it can photobleach during processing. Also, if I use CM-Dil (which is red, I believe), does that mean I cannot do IHC using a red fluorescent secondary (like AlexaFluor 594)? Our sections are formaldehyde fixed and paraffin embedded.

2) Human cells into athymic rats. We are working on a model of xenograft fibroblast transplantation, basically human fibroblasts onto a "scaffold" implanted into an athymic rat knee. How do I track the human cells and differentiate them from rat cells? I assume there are a whole host of proteins that are uniquely human that I can stain for using immunohistochemistry. I've heard human alkaline phosphatase is commonly used for tracking human xenograft implant survival but I'm not certain. Any other ideas for differentiating human from rat and/or implanted cells from host cells?

If anyone understands what I'm saying, PLEASE HELP!!!

-Gabe
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Re: Ex-Vivo Cell Tracking

Postby CrowSan » Jul 27 2012 10:27 am

Hi not sure I can be much help. We have had the same problem of GFP silencing in myoblasts. The promoter (CMV in this case) gets methylated and switches off (a paper we found actually describes this happening in these cells).
Would it be possible to get these cells from younger transgenic rats (or pre-born)? if silencing is a function of division this might help. Don't forget that GFP signal can be destroyed by alcahol fixatives. Thus will be destroyed during wax embedding when the section is dehydrated.
Perhaps another possibility is radio-actively labelling the cells prior to inplantation? Here I am thinking of 35-S Methionine
it has a half-life of 82 days so may last long enough. Also low beta emitter so safe to use on the bench (and should not harm the animals to much). I have no idea what it would do to the cells in the long term though. Detection is normally by exposure to X-ray film (takes about a week for proteins on southern blots but should be shorter if all the proteins in the cell are labelled). Off on holiday now (back in a week). Good luck!
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Re: Ex-Vivo Cell Tracking

Postby mchlbrmn » Jul 27 2012 10:03 pm

Hi. I really don't have expertise to help you, but I'll throw in some ideas in case they stimulate any good thoughts.
You might check out alternate promoters to see if there is on that shows more constitutive robust expression. I don't know if SV40 or EF1a might work better, but you can research alternatives (if you haven't already). Could an inducible work better, or does that just complicate it?

Or, perhaps you would have more success if you try different transfected cell lines. Maybe the methylation Crsn mentions wouldn't occur if the same gene construct integrated into the genome at a different location. After all, the cells must express some proteins. The right promoter in the right locus would have to work. What if you did the GFP transfection, put the transfection cells onto your scaffold, and then later sort the cells to select for cells that express the most GFP in your experimental model, and create some cell lines from those. Oops: am I not allowed to use immortal cells? I see, you start with a transgenic rat. So, in this case I must mean you might have more success with trying different transgenic rats, even if derived from transfection of the same GFP construct, because descendants of different founder transgenic rats will have different integration sites, and copy number at that locus.
Instead of a simple transgenic, the gene might be knocked into a locus/gene known to express in fibroblast/ligament tissue, so the normal methlyation/etc. at that locus would be know not to interfere (hopefully).

Maybe in situ hybridization or PCR might find the genomic GFP in the sections. Oops, I guess FISH is an in situ hybridization, but I guess you can compare other chemical or radioactive probes, and PCR can amplify the signal, if it can apply to your size and situation.

Well, I'm obviously no help, but maybe this will stimulate an idea in someone else.
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Re: Ex-Vivo Cell Tracking

Postby kentboles » Aug 15 2012 4:27 pm

The GFP probes would work in the first experiment, but not the second. Using male cells for the implant into a female host gives you a similar technique for both experiments. There are a lot of protocols and company products for Y chromosome detection. FISH would look nice on histology or you can use qPCR for whole samples / biopsies. Good luck!
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Re: Ex-Vivo Cell Tracking

Postby kmuirhead » Aug 15 2012 4:50 pm

You might consider using PKH26, a yellow-orange emitting membrane dye that has been used to track a wide variety of different cell types in vivo, including long term monitoring of transplanted chondrocytes in a goat articular cartilage model:
Dell'Accio, F et al. Expanded phenotypically stable chondrocytes persist in the repair tissue and contribute to cartilage matrix formation and structural integration in a goat model of autologous chondrocyte implantation. J. Orthop. Res., 21: 123–131, 2003.

PKH26 should be spectrally compatible with your GFP marker and also with an Alexa 594 counterstain. Alternatively you could consider using CellVue Claret, a far red (~650nm) emitting membrane dye, although you would need a red sensitive imaging system to monitor it since the human eye has poor sensitivity at those wavelengths. Both of these dyes decrease in intensity if/as the originally labeled cells divide. To maximize the lenghth of time for which cells could be tracked if they divide in vivo, you would want to do a preliminary titration to determine the maximum concentration of tracking dye that could be used without inhibiting cell growth:
Tario JD Jr et al. Tracking Immune Cell Proliferation and Cytotoxic Potential Using Flow Cytometry. Meth.Mol. Biol. 699: 119-164, 2011.

Full disclosure: in a prior life I worked for Zynaxis Cell Science, Inc., the company that originated the PKH family of tracking dyes, and my current employer (SciGro, Inc.) provides consulting services for Phanos Technologies, Inc. (present owner of the PKH and CellVue dyes) and backup technical support for Sigma-Aldrich (which distributes cell tracking kits containing the dyes).
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Re: Ex-Vivo Cell Tracking

Postby JMinCA » Aug 15 2012 5:29 pm

I have a suggestion and an idea. First, for ways to detect human cells in rodents I suggest that you look at this paper: Cummings, et al. 2005 PNAS, USA., 102(39): 14069–14074 (http://www.ncbi.nlm.nih.gov/pmc/article ... ool=pubmed). Dr. Cummings and colleagues detected human cells implanted into mice by immunocytochemistry for both human nuclear antigen and human cytoplasmic antigen (two different antibodies). This might also work in rats.

As for labeling the fibroblasts, you might try introducing a fluorescently-labeled dextran into the cultures just before implantation, perhaps by electroporation of a fluorescent anionic dextran. Before the use of the carbocyanine dyes (eg. DiI), the dextran-labeling technique was used by developmental biologists to track cell differentiation in embryos. The only problem is the dilution of the label in subsequent generations of daughter cells that results from mitosis, so detection would depend on whether your fibroblasts still undergo a few rounds of mitosis after implantation. However, this technique would label the myoblasts with the color of your choice, and dextran is usually well tolerated (we neurobiologists use fluoro-ruby and fluoro-emerald to label neurons in vivo, and they are pretty fussy cells). I don't know too much about fibroblasts, but it is also possible that they might internalize biotinylated dextran-amine (BDA), which is another tracer commonly used in neurobiology, and is easy to detect.

Good luck.
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Re: Ex-Vivo Cell Tracking

Postby anxiolytic » Mar 12 2014 8:53 am

Hey,

I wonder if you are still working on this?

Using male cells into female hosts you can use in situ probes against the Y chromosome:
http://www.sciencedirect.com/science/ar ... 8X9290137Z
http://journals.lww.com/neuroreport/pag ... e=abstract
http://link.springer.com/article/10.102 ... 8536130578
http://www.ncbi.nlm.nih.gov/pubmed/10735111

You can also design primers against the sex determining region and do a qPCR on the tissue to work out the number of surviving cells.

The only thing I would say is that the in situ isn't so easy, but it does work!

Good luck.
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