March 14, 2011 at 7:50 pm #73570Establishment of clonal myogenic cells in FSHD
Hello, found some info on some research that seems like could benefit not only FSHD but many other muscular dystrophys.
I am not to sure what they are talking about really, so if anyone knows or has a rough idea please explain here in Laymens terms obviously thanks.
Heres the info :
Establishment of clonal myogenic cell lines from severely affected dystrophic muscles:
A hallmark of muscular dystrophies is the replacement of muscle by connective tissue. Muscle biopsies from patients severely affected with facioscapulohumeral muscular dystrophy (FSHD) may contain few myogenic cells.
Since the chromosomal contraction at 4q35 linked to FSHD is thought to cause a defect within myogenic cells, it is important to study this particular cell type, and not the fibroblasts and adipocytes of the endomysial fibrosis, to understand the mechanism leading to myopathy.
Results: We present a protocol to establish clonal myogenic cell lines from even severely dystrophic muscle replaced by mostly fat by overexpression of CDK4 and the catalytic component of telomerase (hTERT), and a subsequent cloning step. hTERT is necessary to compensate for telomere loss during in vitro cultivation, whereas CDK4 prevents a telomere-independent growth arrest affecting CD56+ myogenic cells, but not their CD56- counterpart, in vitro.
Conclusions: These immortal cell lines are valuable tools to reproducibly study the effect of the FSHD mutation within myoblasts isolated from muscles that have been severely affected by the disease, without the confounding influence of variable amounts of contaminating connective tissue cells.
Author: Guido StadlerJennifer ChenKathryn WagnerJerome RobinJerry ShayCharles EmersonWoodring WrightCredits/Source: Skeletal Muscle 2011, 1:12March 15, 2011 at 12:13 pm #79711Re: Establishment of clonal myogenic cells in FSHD
I must admit, I do find these sorts of things very complicated to read and understand at times. It is almost a sub-english language in itself, lol.
Thanks for posting Winged
I'm always the animal, my body's the cage
I blog about nothingness www.amgroves.comMarch 15, 2011 at 12:51 pm #79712Re: Establishment of clonal myogenic cells in FSHD
OMG that is a complicated one, even for me after 8 years studying science!
It sounds like they have developed a model for studying FSH in the lab. I will look into this further and get back to you if I can figure out any more detail than that!
(Research Communications Officer at MDC)March 16, 2011 at 2:44 pm #79713Re: Establishment of clonal myogenic cells in FSHD
I have found a link to the full article. Maybe this will help to understand a bit about what they are trying to do.
Well lets hope it does!March 18, 2011 at 6:42 pm #79715Re: Establishment of clonal myogenic cells in FSHD
So, I’ve read the paper. Basically it is very difficult to grow muscle cells in the lab from biopsies from people with muscular dystrophy because as the muscles waste away they are replaced by fat and scar tissue. This paper outlines a method to isolate the muscle cells and grow them in the lab.
They also made the cells immortal by adding some extra genes. Some cell types like skin cells can be grown in the lab for a reasonable amount of time but unfortunately muscle cells tend to die pretty quickly in the lab making it difficult to do experiments on them. So they genetically engineered the muscle cells so they could be kept alive for a long time (this has been done for lots of different cell types before but I’m not sure about FSHD muscle cells). I’m going to ask one of our researchers if this has been done before and how important this new method is.
For FSH what they really need is a mouse model. Its very difficult to develop therapies without a mouse model to test them on. For example if they develop some kind of gene therapy they need to test it in an animal model to see if it goes to the muscles where it is need and isnt’ toxic.
Kristina.March 19, 2011 at 12:19 pm #79716Re: Establishment of clonal myogenic cells in FSHD
Now why didn’t they just say that LOL
Thanks for reading and translating, much appreciated. You’ve definitely earned two paracetamol and a lie down. If I had sat down and thought about the difficulties of FSHMD research I might have hit on the same things, how to replicate it for testing and how to live test as it were and how to gather the matter in the first place.
Trust me to get one of the awkward types never was comfortable in a pigeon hole
I'm always the animal, my body's the cage
I blog about nothingness www.amgroves.comMarch 19, 2011 at 2:40 pm #79717Re: Establishment of clonal myogenic cells in FSHD
Thanks for taking the time to read this, Kristina.
That has made it a bit clearer. It does sound like that it is a pretty important thing that they have been able to do.
I’m assuming they need to do this so that they can test different drugs and treatments and to see how the cells evolve over a matter of time?
I thought they had a mouse model, can not find to much info on it.
A few years ago a FRG1 mouse model was developed. At that time it was thought that this mouse would be a good model of FSHD. Although it has been of some use, it has proved not to be as good as was thought. Now that more recent research has determined that the DUX4 gene in combination with a particular adjacent chromosome arrangement is the cause of FSHD, a new and more appropriate mouse model must be found or developed.
Apparently an American company has had a donnation from the MDA or someone to help develope a Mouse Model for FSHD.
This was towards the end of last year. The reaserch company is called Harper Lab.
2. “Defining the Tissue and Cell Specificity of the Human DUX4 promoter in Mice”
Scott Harper, Ph.D.
Center for Gene Therapy
The Research Institute at Nationwide Children’s Hospital
The Ohio State University
700 Children’s Drive
Columbus, OH 43205 USA
$50,000 over 1 year
FSHD was formally classified in 1954, and the primary genetic defect, D4Z4 contraction, was identified in 1992, but the pathogenic mechanisms underlying the disease have only recently started to come into focus. One reason for the difficulties in understanding FSHD biology is the lack of a relevant animal model expressing FSHD-permissive D4Z4 arrays. Since animal models, particularly mice, are crucial tools for studying disease pathogenesis and developing potential therapeutics, the absence of an FSHD mouse model is a fundamental problem in the FSHD field. A major goal of the Harper lab is to generate an FSHD mouse model expressing a single FSHD-permissive human D4Z4 repeat, and to use this model to understand the role of the D4Z4-resident gene, DUX4, in FSHD pathogenesis, and develop RNAi therapeutics targeting DUX4.
In preliminary data, supported by previous FSH Society Fellowships to the Harper Lab, we delivered DUX4 to mouse muscle using adenoassociated viral vectors (AAV). DUX4 over-expression in muscle caused myopathy, but DUX4 is generally toxic to many non-muscle cells as well. Thus, we hypothesized that if DUX4 over-expression is an underlying pathogenic event in FSHD, it must be preferentially expressed only in affected muscles. We therefore developed transgenic mice expressing the green fluorescent protein (GFP) gene from the human DUX4 promoter (DUX4p-GFP mice), to determine the tissue and cell specificity of DUX4. In preliminary studies, we observed gross GFP expression in the face, shoulder girdle, and limbs of three independent DUX4p-GFP mouse lines. In this proposal, we will more carefully define the developmental and cellspecific expression patterns of DUX4p-GFP mice, and develop an AAV vector to determine whether a viral-mediated vascular delivery approach can produce the same expression patterns. Ultimately, these studies will be important first steps toward developing an AAV-mediated D4Z4 mouse model.
Specific Aim 1: To define the developmental and cell-specific expression patterns of the human DUX4 promoter in mice. Mounting evidence supports the hypothesis that over-expression of the D4Z4-resident DUX4 gene is an underlying pathogenic event in FSHD. DUX4 is generally toxic to many cell types, and since FSHD is characterized by dystrophy of very specific muscle groups, we hypothesized that DUX4 is preferentially expressed only in affected muscles. Our newly generated DUX4p-GFP reporter mice grossly express GFP in areas that are preferentially affected in FSHD. In this Aim, we will perform a detailed characterization of GFP expression in our DUX4p-GFP mice. These results will help define the expected expression patterns of DUX4, and ultimately increase our understanding about the role of DUX4 FSHD pathogenesis.
Specific Aim 2: To develop an AAV vector-mediated DUX4p-GFP mouse model. Previous endeavors to generate D4Z4 or DUX4 FSHD mouse models using traditional transgenic approaches have been unsuccessful. Although the previous attempts are not published in peer-reviewed literature, the difficulties encountered in generating these models were discussed in abstracts and talks at various scientific meetings over the last several years, including at the FSH Society’s 2008 International Research Consortium and Research Planning Meeting held in Philadelphia, Pennsylvania
Vascular delivery of AAV vectors carrying FSHD-permissive D4Z4 repeats to adult mice may circumvent the early embryonic death or developmental defects arising from germline transmission of D4Z4 repeats using traditional methods. In this Aim, we will test the feasibility of using AAV vectors to drive D4Z4-specific expression patterns in mouse muscle using an AAV.DUX4p-GFP reporter vector.
Heres a link to a list of other reaserch grants and what they are used for, funded in America.
I could not find one for the UK or Europe. Is there anything like this in the UK? If so could who ever point me in the right direction.
Thanks again Kristin, hope it didnt melt your brain to much!
WWMarch 21, 2011 at 5:21 pm #79718Re: Establishment of clonal myogenic cells in FSHD
No problem with the translation, that’s what I’m here for! I heard back from Prof. Jane Hewitt who is funded by us to do FSHD research in Nottingham. She said “This paper describes a protocol for growing muscle cells from muscular dystrophy patients, specifically FSHD patients. This doesn’t tell us anything new about the disease, but does provide cell culture models that are likely to be useful. There is no information in the paper about whether or not this cell line shows any DUX4 expression.”
I will ask her if she is involved in developing a mouse model.
The research we fund is here, with lay summaries. But of course we don’t only fund FSHD:
Jane Hewitt’s project is explained here:
And another one by Peter Zammitt about the involvement of stem cells in FSHD is here:
Other than us, AFM in France is probably the biggest funder of muscular dystrophy research (but their website isn’t very good):
There’s also the Italian Telethon, there’s mention of an animal model in one of their projects but the summary is in italian!
There is also a charity in the Netherlands called the Princess Beatrix Fund which funds a lot of research, but their website is in Dutch.
Hope this helps and keeps you busyMarch 22, 2011 at 12:34 pm #79719Re: Establishment of clonal myogenic cells in FSHD
Step 1 – learn Italian and Dutch
Step 2 – Translate
Awesome work – you are such a mind of information, Thank you.
I'm always the animal, my body's the cage
I blog about nothingness www.amgroves.comMarch 22, 2011 at 6:18 pm #79721Re: Establishment of clonal myogenic cells in FSHD
Thanks for the other info Kristin, it shall keep me busy for a while, but I am always looking for new info so it probably won’t be to long before I am bothering you again!
With regards to a translator, on the new Internet Explorer 9 there is a function where it automatically detects a foreign language on a web page and then translates it into English. Which I think is quite handy if a bit lot lazy!
WWMarch 23, 2011 at 5:01 pm #79722Re: Establishment of clonal myogenic cells in FSHD
I asked Jane Hewitt about mouse models:
“We are not directly making a mouse model, but are interested in the role of DUX proteins in mice. There are several potential mouse models being created, but most are not published yet. Sorry not to be more helpful, but a “true” mouse model is very difficult to generate because of the nature of the mutation. The equivalent region to chromosome 4q35 in the mouse looks very different. I presume the Ohio lab that they refer to is Scott Harper. I know he works on the FRG1 overexpressing mouse (but that’s not a model of FSHD really) and he might be making a DUX4 overexpressing mouse, but again that doesn’t really recapitulate the human disease mutation. Something rather more subtle is needed.”April 19, 2012 at 1:41 am #79724Re: Establishment of clonal myogenic cells in FSHD
o they genetically engineered the muscle cells so they could be kept alive for a long time (this has been done for lots of different cell types before but I’m not sure about FSHD muscle cells). I’m going to ask one of our researchers if this has been done before and how important this new method is.AnonymousInactivePosts: 0Joined: 01/01/1970
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