Characterization of bone marrow-derived mesenchymal stem cell growth on
biodegradable poly-lactic acid films used to facilitate peripheral nerve regeneration
Coffman
1
A.R. ,
Plante
1
K.A. ,
Veach
1
P.M. ,
Zbarska
2,3
S. Sharma
2,4
A.D. ,
Uz
4
M. ,
Mallapragada
2,4
S.K. and
Sakaguchi D.S.
2,3
1 University Honors Program 2 Neuroscience Program 3 Department of Gene@cs, Development and Cell Biology 4 Department of Chemical and Biological Engineering, Iowa State University, Ames, IA 50011 Abstract
Objective: To characterize the growth properties of bone marrow-derived mesenchymal stem
cells (MSCs) cultured on biodegradable micropatterned poly-lactic acid (PLA) porous films
and conduits manufactured with either 10% Pluronic or 10% Salt solution. Once the optimal
PLA film is determined, then PLA films of that production will be used to fabricate conduits
that will be used in future animal studies (rat model) to investigate peripheral nerve
regeneration.
Peripheral nerve injuries (PNI) can lead to serious neurological deficits resulting in sensory/
motor dysfunctions, including paralysis. A major research effort in the Sakaguchi lab is the
development of experimental strategies to facilitate nerve regeneration. Autologous nerve
transplants including Schwann cells (SCs) are considered the gold standard for treatment of
PNI. SCs are peripheral glia forming a myelin sheath around axons of motor and sensory
neurons. SCs also secrete trophic and growth factors which promote neural regeneration.
However, SC harvesting requires an additional surgery and sacrifice of a donor nerve that
may result in donor site morbidity. Due to these limitations, alternative cell sources are being
investigated.
MSCs are multipotent somatic cells that are easily isolated and maintained in culture, and
may be used for autologous grafting procedures. Furthermore, under the appropriate
conditions MSCs can be transdifferentiated into SC-like phenotypes (tMSCs). These cells
resemble typical SCs morphologically and molecularly. Identification of the transdifferentiated
MSCs (tMSCs) was based upon immunolabeling with antibody markers used to identify SCs,
anti-S100β and anti-p75NTR.
The focus of this work is to characterize the growth properties of tMSCs cultured on
micropatterned PLA porous films and conduits manufactured with either 10% Pluronic or 10%
Salt. It is expected that porous films will be more permeable for nutrients to move in and
waste products to move out of conduit lumen while implanted in vivo.
Culturing and Transdifferentiation of MSCs into a
Schwann-cell like Phenotype
DAPI
tMSCs_PLA_s100β_pat
tMSCs
S100β
Phalloidin
Culturing of tMSCS on 10% Pluronic and Salt Poly-­‐lac7c Acid Micropa:erned Conduits Merged Image Figure 4. Transdifferentiated MSCs
expressed SC-marker proteins S100β
(red) staining on patterned PLA substrates.
Phalloidin (F-actin) and DAPI (nuclei)
staining is shown in green and blue
respectively. 1
3
DIV
uMSCs_PLA_s100β_pat
uMSCs
• MSCs on patterned substrate aligned themselves in the
direction of the microgrooves while cells on smooth substrates
were randomly oriented.
tMSC uMSC tMSC uMSC tMSC uMSC tMSC uMSC
Pa
Sm Sm Pa Pa Sm Sm
Pa
• MSCs subjected to transdifferentation expressed SC-marker
s100β
p75NTR
proteins S100β and p75NTR in significantly higher
Figure 5. Percentage of S100β and
percentages as compared to the control MSCs (uMSCs) on p75NTR labeled cells. Error bar
represents standard error of the mean.
both PS and PLA substrates.
N=3. S100β and p75NTR labeling of the
• Substrate topography did not significantly affect the
tMSCs was significantly greater (p ≤
percentage of tMSCs suggesting that micropatterning does 0.05) than the uMSCs for all substrate
types. Pa: Pattern, Sm: Smooth.1
1
not cause any decrease in the level of transdifferentiation
Culturing of tMSCS on 10% Pluronic and 10% Salt
porous micropatterned PLA films
Materials and Methods Micropatterned biodegradable porous
polymer films and conduit fabrication:
• 10% biodegradable poly-lactic acid (PLA)
• 10% Pluronic: 10 mg of Pluronic F-127
polymer
1 mL of 10% PLA solution
400 µL of Pentane
•  10% Salt:
10mg of Salt
1 mL of 10% PLA solution
400 µL of Pentane
•  Pour solution on wafer and use spin coater
•  Place in to the water bath over night
Results
Results
x 7
DIV
Figure 8. tMSCs shown on 10% Pluronic Micropatterned conduits. Cells were stained red with RhPh and blue with DAPI fluorescent
dyes 36
HIV
5
DIV
Figure 9. tMSCs shown on 10% Salt Micropatterned conduits. Cells were stained red with RhPh and blue with DAPI fluorescent
dyes.
3
DIV
Figure 1. Images of PLA micropatterned film and PLA 10%
Salt conduit (conduit dimensions: L=12 mm, ID= 1.8 mm,
ED=2.2 mm).
Isolation and culturing rat MSCs:
• MSCs were isolated from the femora and tibia
bone marrow of Brown Norway rats
• MSCs were characterized using positive and
negative MSC antibody markers
Figure 2. Scanning Electron Microscope (SEM) images of
• MSCs were genetically modified to express
10% Salt and 10% Pluronic porous micropatterned films.
Green Fluorescent Protein (GFP)
Groove 11-13 µm, mesa 16-18 µm and depth 3-4 µm.
Transdifferentiation of MSCs:
Isolated MSCs were incubated in 3 transdifferentiation media (TDMs):
• TDM1 (1 day): αMEM supplemented with 1 mM β-mercaptoethanol
• TDM2 (3 days): αMEM, 10% FBS, 35 ng/ml all-trans-retinoic acid
• TDM3 (8 days cultured on substrates): αMEM, 10% FBS, 14 µL forskolin,
5 ng/mL platelet derived growth factor, 10 ng/mL basic fibroblast growth
factor, 200 ng/mL heregulin β1
Culturing on PLA Films and inside of Conduits
•  Films and conduits were covered with rat laminin solution 1 mg/mL for 12 hours
•  tMSCs were seeded on films with cell density 6000 cells/cm2
•  tMSCs were seeded in conduits with cell density 60,000 cells/conduit
•  tMSCs on films or in conduits were cultured in TDM3 for 10 days
Staining and Image Analysis
•  Cells on films and in conduits were fixed in 4% paraformaldehyde for 20 minutes at
multiple time points during 10 days of culturing
•  DAPI and Rhodamine Phalloidin fluorescent stain were applied for 30 minutes
•  Staining was visualized using fluorescent microscopy
7 DIV
Figure 6. tMSCs cultured on a 10% Pluronic micropatterned porous films stained after 3 and 7 days in vitro (DIV). Cells were
stained with Rhodamine Phalloidin (RhPh) (red) and DAPI (blue) fluorescent dyes. Cells were fixed after 3 and 7 days in vitro.
(DIV)
3
DIV
6
DIV
Figure 7. tMSCs cultured on a 10% Salt micropatterned porous films stained after 3 and 7 days in vitro (DIV). Cells were
stained with Rhodamine Phalloidin (RhPh) (red) and DAPI (blue) fluorescent dyes. Cells were fixed after 3 and 6 days in
vitro. (DIV)
•  GFP expressing tMSCs adhered, aligned, and survived on 10% Salt and 10% Pluronic PLA
films.
•  No decrease in the number of tMSCs was observed one week after plating.
•  The experiment with cells cultured on 10% Pluronic PLA films was repeated 3 times. In one
experiment the cells formed cell spheres instead of aligning on the film.
•  GFP expressing tMSCs adhered, aligned, and survived on 10% Salt and 10% Pluronic PLA
conduits.
•  No decrease in the number of tMSCs was observed one week after plating
•  The experiment with cells cultured on 10% Pluronic PLA conduits was repeated 3 times. In one
experiment the cells formed cell spheres instead of aligning on the film.
Conclusions
•  MSCs can be transdifferentiated successfully to Schwann-like cell phenotypes on both patterned
and smooth PLA films.1
•  Topography of polymer substrates strongly influenced the morphology and growth of the uMSCs
and tMSCs. Cells aligned in the direction of the microgrooves when grown on micropatterned
substrates.1
•  tMSCs adhered and aligned on 10% Pluronic and 10% Salt PLA films and conduits.
•  No decrease in the number of tMSCs were observed one week after plating.
•  We recommend to use 10% Salt PLA micropatterned conduits for future animal research due to
the consistency of results.
Future Research
•  Repeat experiment with culturing GFP expressing tMSCs on 10% Salt PLA Micropatterned Films
and 10% Salt PLA Micropatterned Conduits to support results.
•  Determine the effects of biodegradation of 10% Salt PLA conduits on cell growth properties during
a prolonged period of time (4+ weeks)
•  Preseed 10% Salt porous micropatterned PLA conduits with GFP expressing tMSCs and then
implanted in rats to facilitate peripheral nerve regeneration
Acknowledgements
1 Zbarska, S., Sharma A.D., Mallapragada S.K., and Sakaguchi D.S. “A peripheral nerve
regeneration strategy using a combination of bone marrow-derived stem cells
transdifferentiated into Schwann-like cells and micropatterned biodegradable polymer
conduits.” Society for Neuroscience. Iowa State University. Washington D.C. 18 Nov. 2014.
Poster Presentation.
Members of the Project: Morgan Bobb, Rachel Cheng, Nhu-Ngoc Doan, Daniel Stroud, Sara
Stuedemann, Aygul Parpucu, Brian Thielen, and Jose Suarez.
The members of the Sakaguchi Lab.
Thanks also to the University Honors Program.
Financial support provided by: We are grateful to the Iowa State University Foundation for
support of this project through the University Honors Program and Stewart Research Grants.
Project financial support from The US Army Medical Research and Material Command
#W81XWH-11-1-0700; Iowa State University; The Stem Cell Biology Fund