Mesenchymal Stem Cells - Interest Group
MSC BIO-FORUM
Giving a Voice to MSC Researchers
I'm disappointed because I've tried more than once, to infect BT549 cells (breast
cancer mesenchymal cell line) with my cDNA in pBabe-puro vector using Nolan's
lab protocol and another protocol gave me from another lab.

Every time my protein of interest is not expressed.

I really don't know what to do. Anyone have suggestions?

Thanks,
Dr. Andrew
POSTED BY:
Dr. Andrew

Feb 16 201
0,
08:19 AM
Dr. Andrew - Did you make a quantitation of infectivity of your
packing-cells-supernatant ?

One of my colleague who works on endothelial cells told me that cells exist that
are strongly resistant to infectivity. Are there references of infection of your cells?
Did you check at the ATCC about this type of information?
POSTED BY:
Dr. Matold

Feb 17 201
0,
11:10 AM
Anybody experience with mouse bone marrow culture?

I have isolated mouse bone marrow cells from tibia and femur of hind legs using
DMEM low glucose or Iscove’s Modified eagles medium (IMDM) but so far I have
not been able to maintain them in culture (incubator 37C and 5% CO2). I used
the following culture/maintenance mediums:

DMEM low glucose + 20% FBS, 0.01% PS
IMDM + 20% FBS, 0.01% PS
DOM medium (IMDM + 15% heat inactivated FBS + 15% heat inactivated Horse
Serum + 1uM hydrocortisone + 0.1mM beta-Mercaptoethanol + 200mM L-
Glutamine + 0.5% PS)
alpha-MEM + 10% FBS + 1% L-glutamine, 1% PS

The mBM cells are either fresh or frozen in cryomedium (40% DL, 40% FBS and
20% DMSO). In the entire mediums above, there are a portion of the token cells
which adhere to the flask surface (Falcon Primaria 25 cm2) while the other portion
is floating cells. Some of those adherent cells have fibroblast morphology, some
are compact round shape and others of strange morphology!!

I am seeking to expand the adherent mBM cells up to 5-8 passages. In the
above culture medium, there are a few colony-forming-units that appear but the
flask never reaches confluence. 30% confluence takes 3 weeks or more, for 1
million cells per ul culture medium seeded.
Help would me very much appreciated!!
POSTED BY:
Dr. Demniz

Jan 29 201
0, 10:09
AM
Hi Demniz - I know a guy around here who successfully isolates and cultivates
bonemarrow-derived macrophages from mice. If you're interested, I can ask him
what media an conditions he uses...

Mike
POSTED BY:
Dr. Mike Albright

Jan 29 201
0, 04:15
PM
I am suffering with non-viral transfection of human mesenchymal stem cells. I
have tried several transfection reagents including Lipofectamine 2000,
Lipofectamine, Effectine, Gene Juice, Fugene 6, calcium phosphate, and also tried
electroporation (Bio-rad Gene Pulser II and Amaxa Nucleofactor+Human MSC kit)
but none of them worked.

I have read few papers that successfully transfect MSC with Lipofectamine, calcium
phosphate, Amaxa Nucleofector and Eppondorf Multiporator. But I failed to see
any glowing cells (my construct is pEF-X-IRES-hrGFP) by using these method
(expect Eppondorf Multiporator, I haven't tried it yet). Should I tried more
different conditions with these have-been-succeeded methods? Or non-viral
transfection of MSC is really so tricky thus I should switch to retrovirus systems or
so? (Btw, what are the advantages and disadvantages of adenovirus, AAV, and
retrovirus? I do not know how to choose...)  Is there any one having experience
with it and can give me some advice? I would very much appreciate it.
POSTED BY:
Dr. Selline

Jan 08, 201
0,
08:55 AM
Hi Dr. Selline,
could it be that your vector is not activated when transfected? Are you sure that it
works, did you see expression and glowing with it somewhere else?
POSTED BY:
Dr. Cantolne

Jan 09, 201
0,
08:35 AM
I have transfected my construct in 293T and seen it glowing.
Is that what you meant? Thanks!
POSTED BY:
Dr. Selline

Jan 09, 201
0,
10:35 AM
Hi, Yes that is what I meant. OK, so have you tried to transfect these cells before,
with someother vector? Just to make sure that it's a problem with transfection...
POSTED BY:
Dr. Cantolne

Jan 09, 201
0, 3:46
PM
We have been using Epoch Biolabs' GenCarrier-1 and -2 (www.epochbiolabs.com)
for primary Human Adult pancreatic cells and putative pancreatic progenitor (may
be similar to mesenchymal stem cells) with 20-30% efficiency which exceed any
other transfection reagents we've been tried (same as the ones you tried). Most
importantly, the transfected cells still growing without any cytotoxic signs. You
may consider it.
POSTED BY:
PostDoc Bradford

Jan 10, 20
10,
11:04 AM
True, mesenchymal cells are not easily transfected - the efficiency is about
20%~30%, I have transfected rat mesenchymal stem cells with p-EGFP-n3 by
lipofectamine2000, with the efficiency at about 30%.
So I don't think it's because of lipofectamine2000, but be sure your vector or cell
are working well.
Good luck.
POSTED BY:
Kingsbrawn

Jan 11, 201
0,
08:08 AM
I have had very good experience with the amaxa nucleofector and the gene
transfer into hMSC, 40-70% are absolutely no problem. I think the main problem
is the vector, although your construct does work in 293 cells, it could be that the
expression pattern in primary cells is different.

A friend of mine, Dan Gazit from Israel, is promoting this technology for non-viral
tranfection into human MSC on his homepage. He is an opinion leader in tissue
engineering, perhaps you can find more clues on his hompage. Just search with
google : Dan Gazit, "mesenchymal stem cells."
Best Wishes,
Peter
POSTED BY:
Dr. Peter Dipple

Jan 13, 201
0, 5:02
PM
Just to share with everyone, I've tried several cell transfection reagents on
primary pancreatic islet cells, endothelial, mesenchymal and hepatocye progenitor
(stem) cells. Among the reagents from the vendors like Invitrogen, Roche,
promega, Stratagen, Gene Therapy Systems, Epoch Biolabs...., i'm surprised to
find that GenCarrier-1 (Epoch Biolabs, $180/mL) gave the best and most
consistent results with highest transfection efficiency, although I did not know this
company before and just tried it by the word of mouth from my friend at FCCC
(Fox Chase Cancer Center).
POSTED BY:
Dr. Adams

Jan 05, 201
0, 5:02
PM
I had done stable transfection of SP2/0 cells using antibiotic G418. What is the
recommended concentration of that? Also how to plot dose response curve and
how to select transformed cells? Thanks for any input you can provide.

Shilpi
POSTED BY:
Dr. Shilpi

Jan 05, 2009, 5:02
PM
I have currently started working with human mesenchymal stem cells (hMSC).  I
am having trouble with the cells lifting in culture (some) and when I try to fix
them (most) before using a von Kossa stain.  Any ideas how I could:

a) make a stronger matrix, or
b) use a different, stronger fixative that agrees with silver nitrate staining?
POSTED BY:
Dr. Sheas

Jan 02, 201
0, 1:01
PM
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Is anyone familiar with use of LUMENESC for detection and measurement of
mesenchymal stem cells? It is my understanding that it uses the same ATP
bioluminescence signal detection system as HALO, but I don't know to much else
about it.  I work with assessing toxicity to the MSC system so think it would be
appropriate for my work. Any feedback would be appreciated. I'm looking to
streamline my work.
POSTED BY:
Mossner, Ph.D.

Feb 17 20
10, 4:56
PM
Yes, you can easily implement the LUMENESC Platform for standardized analysis
of a MSC / CFC-F population or differentiating populations. You'll save a lot of
time compared to growing CFC-F in Petri dishes and staining in order to count
under a microscope. Btw, HemoGenix offers a kit is to detect apoptosis in the MSC
system. Might be worth taking a look at that given your line of work.
Best, Richard
POSTED BY:
Serfold

Ma
r 15 2010, 8:11
AM
Any preferences on working with rat vs. mouse mesenchymal stem cell
populations? There seem to be more commercial options for rat MSC products,
but I'm wondering if that's because of ease of use or patent restrictions (or
substantially similar factors). My  experience to date has been with mouse MSC
populations, but it seems that my best options for commercial media, kits, etc. is
designed for compatability with rat MSC. Thanx for your thoughts.
POSTED BY:
Zignlod

Feb 11 201
0, 3.:32
PM
1. It is my understanding that the patent environment is slightly less restrictive
for rat mesenchymal stem cell, at least compared to human mesenchymal stem
cells.

2. I think the main advantage of rat MSC is the handling advantage. Easier
growth properties, and in my experience, best for inducing differentation into
downstream lineages.
POSTED BY:
Nienfield

Feb 13 2010,
10:28 AM