Neurogenic
Peripheral Blood-Derived Adult Stem Cells
Laura
Pasieniuk and Elmer M. Price
Dalton Cardiovascular Research Center;
Department of Biomedical Sciences, College of Veterinary Medicine, University of Missouri-Columbia
ABSTRACT
The therapeutic
use of stem cells is a promising avenue emerging in science. Embryonic sources were the first known
providers of stem cells, yet in recent years adult sources of stem cells have
been discovered in bone marrow and peripheral blood. Peripheral blood draws
from green fluorescent pigs have yielded stem cells with mulipotent
capabilities. These cells, known as peripheral blood derived multipotent adult
progenitor cells (PBD-MAPCs), have been shown to differentiate into five
different cell types including adipocytes, osteoblasts, endothelial cells,
smooth muscle cells and most interesting, neural cells. PBC-MAPCs are maintained in an
undifferentiated state as spheroids which possess the ability to change into
different cell types depending on the signals they receive. The goal of this project is to define these
factors or mechanisms that define neurogenesis, particularly into oligodendrocytes
and dopaminergic neurons. This process
begins by plating PBD-MAPCs on MatrigelTM and feeding
the cells media containing fetal bovine serum.
When exposed to these conditions PBD-MAPCs express several different
neural markers including NeuN, TH, MAP 2a+b, MPB, GFAP, and beta-3
tubulin. Our goal is to explore the
effects of BMP-4, Sonic hedgehog (Shh), BDNF, NGF, Netrin-1, Semaphorin 3a,
and FGF8. The analysis of these
specific factors will allow us to evaluate the expression of specific neural
subtypes for therapeutics in areas such as spinal cord injury and Parkinson’s
disease.
1. PBD-MAPC Isolation. Peripheral blood mononuclear cells were
isolated from GFP-swine using density gradient centrifugation. Cells were mixed with semi-solid media
containing growth factors and cytokines (selected for their role in stem cell
growth). Colonies (100-300) were picked
and expanded in Primordial Media.
Typically, 1-3 colonies continue to proliferate. We have generated 5 lines of PBD-MAPCs from
2 animals.
2. PBD-MAPCs Grow as Spheroids. These structures are reminiscent of embryoid
bodies or neurospheres. These are
passaged every 7-10 days using collagenase and dispase.
3. Dispersal of PBD-MAPC Spheroids. Samples were incubated with collagenase IV
and dispase for 30 min. (37o). This
procedure dissociates spheroids into individual cells, which are seeded 1:3
into fresh dishes. Cells typically
re-grow as spheroids.
Specific
Goals
5. The goal
of our study is to determine the factors required in neurogenesis of PBD-MAPCs. Differentiated cells’ phenotypes will be
analyzed for specific neural markers using immunoblots. Specifically we will be testing the factors
that determine the differentiation of cells into oligodendrocytes and
dopamingeric neurons
SUMMARY
-We have developed neurogenic progenitor cells from adult blood cells.
-These cells rapidly
differentiate into cells that express molecular markers consistent with neural cell types.
-PBD MAPC-derived neurogenic
cells appear to
attenuate spinal cord injury in a rat model.
-Netrin-1 is responsible for
neural differentiation
and will be used to pre-induce cells for spinal cord injury therapy.
Future
directions include additional marker analyses and cell transplantation into
rat models of Parkinson's disease and spinal cord injury. We are extending these studies to humans and
this will pave the way for patient-specific or haplotype matched adult stem
cells for transplant procedures.
Previous Work
4. Confocal
Microscopy performed on PBD-MAPCs introduced to different factors indicated
different conditions induced neurogenesis.
These cells are inherently intensely fluorescent since they are derived
from GFP+ swine.
When grown on #1 glass coverslips and stained with the different
antibodies, cells showed fluorescence for anti-MAP 2ab, anti-bIII
tubulin, anti-tyrosine hydroxylase, and glial fibrillary acid protein. The probed antibody fluoresced red in these
samples with GFP+ (green cells).
Materials and
Methods
6. PBD-MAPC
spheroids were dissociated using a 0.1% Collagenase and 0.1% Dispase solution
at 37o C for 1.5 hrs. During this time cells were
vortexed and titruated three times.
After dissociating and washing the cells, the desired number of cells
was counted by manual methods and plated on a 12 well plate containing 1:1
VSM/EBM-2 media with different neurogenic factors. Each neurogenic factor was mixed to a final
concentration of 30 ng/mL. Media was
changed every 4-5 days and cells were harvested after 10-14 days.
After cells had
reached desired maturation, they were harvested for immunoblots. Cells were washed twice with PBS. 250 ul of Laemmili were added and incubated for 15
minutes at 72o C. 20 ml
of solution was loaded on a 4-12% gradient gel and run at 200 volts for 55
minutes. While the gel was running, a
PVDF membrane was prepared. After 55
minutes in the running buffer, the gel was transferred at 200 mA for 1.5
hrs. After the transfer, the membrane
was blocked in a 5% milk TBS/Tween-20 solution for one hour at room
temperature. This solution was decanted
and the desired antibody was added to the membrane in a 5% milk solution which
incubated at 4o C overnight.
Blots were rinsed with 1X TBS/Tween-20 solution for 10 minutes a
minimum of five times. Secondary
antibody was then added via a 5% milk solution and incubated will rocking at
room temperature for one hour. Blots
were then washed again with 1X TBS/Tween-20 solution. ECL reagent was added to the blots with a 60
second agitation period. Blots were
quickly dabbed onto filter paper, wrapped in cellophane and placed into the
cassette. Exposure time varied
according to the antibody used.
30 ng/mL
Process
growth
Fibroblast
Growth Factor (FGF-8)
30 ng/mL
Process
growth
Semaphorin
3a
30 ng/mL
Process
growth
Netrin-1
30 ng/mL
Neurons
Neutrophic
Growth Factor (NGF)
30 ng/mL
Neurons
Bone
Derived Neutrophic Factor
(BDNF)
30 ng/mL
Dopaminergic
neurons/ oligodendrocytes
Sonic
Hedgehog (Shh)
30 ng/mL
Dopaminergic
neurons/oligodendrocytes
Bone
Morphogenic Protein
(BMP-4)
Concentration
Used
Proposed
Differentiation
Factor
Neural Differentiation Factors
10. Immunoblot of PBD-MAPCs probed with MAP 2ab. Lane 1- control, Lane 2- BMP-4, Lane 3- Shh, Lane 4- Shh & BMP-4, Lane 5- BDNF, Lane 6- NGF, Lane 7- BDNF & NGF, Lane 8- Netrin, Shh & BMP-4, Lane 9,- Netrin, BDNF & NGF. Results show a band in Lanes 1, 4, 6, 7, 8, 9 near the top of the picture.
This band is very faint in lanes 1, 4, & 6 and intense in lanes 8 & 9.
The presence of this band indicates the presence of MAP 2ab in the prepared samples. MAP 2 ab is a factor expressed in mature neuronal cells.
The presence of this band indicates that these cells are now differentiated neural cells. Due to the fact Netrin-1 is present in lanes 8 & 9 we conclude that this neurogenic factor may be involved in neural expression.
8. Recovery of Voluntary Motor Skills in SCI-Transplanted Rats. Images are 4 weeks post-operative and are of
2 different animals. Panels A1-A3 are three pictures in rapid succession of a rat rearing to peer over
the edge of the open field arena. The
animal clearly brings this legs to a nearly plantar-down position and attempts
to push with his hind legs. This is a
highly reproducible movement. Panels B1-B3 are 3 pictures in rapid succession of a different animal during a BBB
open field locomotor assay. While
scoring very low, the animal clearly attempts to utilize his hind limbs
(B1-B2-B3 appear to be the coordinated movement of a single step).
7. Spinal Cord Transection. A.
Photograph showing the exposed spinal cord at T10; B. Same field as A, after
removal of about 2-3 mm of spinal cord (arrow). Stem cells were inserted into this void
9. Transplanted PBD-MAPCs Survive 4 Weeks Post-Op. Cells were pre-induced with BMP-4 in a
Matrigel/Fibrin plug, which was transplanted into transected spinal cord
(Figure 7). Following 4 weeks (where
motor function was beginning to return), one animal from the cohort was
sacrificed, perfusion fixed in 4% PFA and the spinal cord removed (4 cm total,
2 cm on either side of the transplant).
The cord was dissected longitudinally and observed via fluorescence
stereomicroscopy at the indicated magnifications. Panel
A is about 5 mm rostral from the
transplant, Panels B-E are from the transplant site, and Panel F is 5 mm caudal to the transplant site.