Note: Descriptions are shown in the official language in which they were submitted.
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OLIGODENDROCYTE CELL CULTURES AND
METHODS FOR THEIR PREPARATION AND USE
This application claims priority to U.S. Provisional Application No.
60/161,125, filed October 22, 1999, the entirety of which is incorporated by
reference
herein.
Pursuant to 35 U.S.C. ~202(c), it is acknowledged that the U.S.
Government has certain rights in the invention described herein, which was
made in
part with funds from National Institute of Health Grant Numbers NINDS,
NS01931,
NS36265 and NS37927.
FIELD OF THE INVENTION
This invention relates to the field of cell culture methods and methods
for treatment of diseases of the central nervous system.
BACKGROUND OF THE INVENTION
Various scientific and scholarly articles are referred to throughout the
specification. These articles are incorporated by reference herein to describe
the state
of the art to which this invention pertains.
Recovery in central nervous system (CNS) disorders is hindered by the
limited ability of the vertebrate CNS to regenerate lost cells and re-
establish
functional connections. In many CNS disorders, including multiple sclerosis,
stroke,
spinal cord injury and other trauma, demyelination of intact axons is an
important
factor contributing to loss of function. Previous studies suggest that
substantial
recovery of function might be achieved through remyelination of otherwise
intact
neural pathways. As a therapeutic modality, functional recovery through
remyelination may prove much easier to achieve than recovery via regeneration
of
severed axons, where appropriate synaptic connectivity must also be re-
created.
Transplantation approaches utilizing cellular bridges, fetal CNS cells,
fibroblasts expressing NT-3, hybridoma cells expressing inhibitory protein
blocking
antibodies, or olfactory ensheathing glial cells into the acutely injured
spinal cord has
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produced axonal regrowth or functional benefits. Transplants of rat or cat
fetal spinal
cord tissue into the chronically injured cord survive and integrate with the
host cord,
and may be associated with some functional improvements. In addition, rats
transplanted with fetal spinal cord cells exhibited benefits in some gait
parameters,
S and the delayed transplantation of fetal raphe cells can enhance reflexes.
Neural
progenitors isolated from the adult CNS differentiate into neurons and glia
after
transplantation into brain (Gage, et al., (1995) Proc. Natl. Acad. Sci. 92,
11879-
11883), and differentiate into oligodendrocytes and astrocytes after
transplantation
into spinal cord.
Neural transplantation studies have been limited by ethical
considerations and a lack of a reliable source for undifferentiated
pluripotent cells. In
vivo differentiated neural cells cultures are problematic because may contain
genetically abnormal cells. Ideally, in vitro neural cell cultures would
provide a better
source of materials for transplantation, and other commercial and research
purposes.
ES cells provide a partial solution to the problems encountered with in
vivo derived neural cells because they are genetically normal, totipotent,
capable of
indefinite replication (Suda, Y et al, (1987) J. Cell. Physiol. 133, 197-201)
and have
been derived from several vertebrate species including mice (Evans & Kaufinan,
(1981) Nature 292, 154-156; Martin, (1981) Proc. Natl. Acad. Sci. USA 78, 7634-
7638) and humans (Thomson, et al., (1998) Science 282, 1145-1147; Shamblott,
et
al., (1998) Proc. Natl. Acad. Sci. USA 95, 13726-13731). ES cells are also
among the
most flexible stem cell for genetic engineering. For example, the production
of
double gene allele knockouts in single ES cells has been accomplished (Wilder,
et al.,
(1997) Dev. Biol. 192, 614-629; Hakem, et al., (1998) Cell 94, 339-352).
In theory, ES cells can generate all cell types and preliminary work has
shown that differentiation into oligodendrocytes is possible (Fraichard, et
al., (1995)
J. Cell Sci. 108, 3181-3188; Dinsmore, et al., (1996) Cell Transplant. 5, 131-
143;
Brustle, et al., (1997) Proc. Natl. Acad. Sci. USA 94, 14809-14814; Brustle,
et al.,
(1999) Science 285, 754-756). However, simple and reliable methods for
producing
and enriching oligodendrocytes have not been developed.
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SUMMARY OF THE INVENTION
The present invention provides a method to treat spinal cord
degeneration through the transplantation of in vitro differentiated cultures
of neural
cells. The inventors, through their superior appreciation of the problems of
spinal
cord transplantation techniques, have recognized the advantages of in vitro
cultured
cells. Furthermore, the inventors have discovered that oligodendrocytes are
the
critical cells required for transplantation. In accordance with the discovery
of the
importance of oligodendrocytes, a method to make an enriched culture of
oligodendrocytes is provided. The highly enriched oligodendrocyte culture of
the
invention can be used to particular advantage in the method to treat spinal
cord
degeneration of the invention to increase the myelination of axons in
degenerated
spinal cord tissues.
According to one aspect of the present invention, a method to make a
cell culture highly enriched in oligodendrocytes is provided. In one preferred
embodiment, the method uses retinoic acid differentiated 4-/4+ stage EB cells.
In
another preferred embodiment, the method uses preconditioned oligosphere
media,
thereby forming an intermediate cell type referred to herein as
"oligospheres".
Another aspect of the invention features a cell culture enriched in
oligodendrocytes. The oligodendrocyte-enriched culture may comprise from about
20% to about 99% oligodendrocytes in the population. In a preferred
embodiment,
the culture is made by the inventive method described herein.
According to another aspect of the invention, a method is provided for
using an in vitro differentiated culture of neural cells to treat degenerated
CNS tissue.
This method comprises the steps of transplanting in vitro differentiated
neural cells
into the spinal cord of a patient in need of such treatment, and allowing the
transplanted cells to replace damaged or missing tissues. In a preferred
embodiment,
the neural cell culture of this method comprises the enriched oligodendrocyte
culture
of the invention.
Other features and advantages of the present invention will be
understood by reference to the drawings, detailed description and examples
that
follow.
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BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1. BrdU labeled ES cell-derived cells 2 weeks after
transplantation. Mean~SEM BrdU labeled nuclei per one mm segment in
longitudinal
sections (n = 11 rats, 3 sections per rat).
Fig. 2. ES cell-derived cell transplantation improved behavioral
recovery. Fig. 2a) Closed circles, ES cell transplant group; open circles,
vehicle
treated group (n=11 per group, mean ~ SEM). *Difference at P < 0.05 vs.
control at
same time point (repeated measures ANOVA with Tukey's test). Fig. 2b) Similar
experiment comparing transplantation of ES cells (closed circles), vehicle
(open
circles), or adult mouse neocortical cells (closed diamonds) (n = 6 per
group). The ES
cell transplantation group differed from both control groups at the P<0.05
level.
Arrows indicate transplantation.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides compositions and methods to
regenerate the tissue of the central nervous system. Surprisingly, it has been
discovered that the transplantation of a mixed population of in vitro
differentiated
neural cells into a damaged spinal cord is associated with improvements of CNS
function. It has further been discovered that a preparation of a highly
enriched culture
of in vitro differentiated oligodendrocytes is unexpectedly effective in
restoring CNS
function by transplantation.
Thus, in accordance with the invention, a method to make a highly
enriched culture of oligodendrocytes is provided. These highly enriched
cultures of
oligodendrocytes can be produced from ES or other totipotent cells by
generating an
intermediate stage of floating cell groups termed "oligospheres". Oligospheres
primarily contain an early form of oligodendrocyte progenitor, and can be
produced by
culturing a mixed population of in vitro differentiated neural cells in a
preconditioned
oligosphere medium, described in greater detail below. The resulting highly
enriched
culture of oligodendrocytes is a novel cell culture that will have many uses
in research
as well as transplantation therapy.
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As described in Example 1, mouse derived embryonic stem (ES) cells
induced to differentiate into a mixed population of neural cells by retinoic
acid were
transplanted into physically and chemically injured spinal cords of rats. The
transplanted mouse cells differentiated into primarily oligodendrocytes in the
spinal
tissue and the rats had improved hind-limb functionality as compared to the
control
group. The use of ES cultures was surprisingly superior to the use of in vivo
derived
cells in the speed at which the transplanted cells migrated into the damaged
spinal
cord tissue and degree of remyelination.
A method to culture a highly enriched population of oligodendrocytes
from mixed population of neural cells was developed and optimized, as
described in
Examples 2 and 3. The method utilizes a novel culture medium that selectively
encouraged the growth of an intermediate class of cells termed "oligospheres",
containing primarily immature and mature oligodendrocytes. This enrichment
method
is based on the unexpected simple concept of using the preconditioned media
from
existing oligodendrocyte cultures to selectively induce the division of
oligodendrocytes in a mixed population to neural cells.
When this enriched oligodendrocyte culture was transplanted into the
spinal cord of shiverer mice, which lack myelin basic protein, the introduced
cells
oriented with native oligodendrocytes and myelinated the native axons. The
enriched
oligodendrocyte culture was surprisingly more effective than the retinoic acid
differentiated ES cells in the rate of cell migration and myelination in the
spinal cord.
The present invention provides a method of therapy for degenerated
CNS tissues. The method comprises the step of transplanting in vitro
differentiated
neural cells into the site of injury and allowing sufficient time for the
introduced cells
to replace missing or defective cell types. This method is particularly
effective when
a highly enriched population of oligodendrocytes is used for the
transplantation.
While not limiting the way in which the transplanted cells benefit CNS
function to any one theory, it appears that the transplanted oligodendrocytes
act by
remyelinating damaged axons. In many injuries to the spinal cord, the axons
remain
intact but are rendered useless due to the loss of their myelin sheath.
Oligodendrocytes in the CNS (and Schwann cells in the peripheral nervous
system)
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wrap the axon of a nerve in a myelin sheath and prevent current from leaking
across
the axonal membrane. The insulating function of the myelin allows the action
potential to move faster along the axon and to use less metabolic energy. When
the
myelin sheath is absent from the majority of axons in a region due to injury,
genetic
mutation or disease, signals cannot be carried up and down the spinal cord,
resulting
in loss of muscular control and/or sensory signals. Remyelination may
therefore be
able to restore nervous system function in some situations.
Accordingly, the use of in vitro differentiated oligodendrocytes is
particularly useful in situations involving loss of myelination. In
particular, this
method of therapy is contemplated to be useful for treating multiple
sclerosis,
Alzheimer's Disease, leukodystrophies, cerebrial palsey, stroke, cardiac
arrest and
CNS trauma, among others.
To facilitate performance of the aforementioned therapeutic methods,
the inventors have determined how to manipulate cultured pluripotent cells to
produce
a culture of differentiated neural cells that is highly enriched in
oligodendrocytes.
Accordingly, an oligodendrocyte-enriched cell culture is featured in
accordance with
the present invention. It has been discovered that such an oligodendrocyte-
enrichced
culture comprising as few as about 20% oligodendrocytes in the population is
suitable
for use in the therapeutic methods of the invention. However, the inventors
have
devised methods for producing a culture comprising up to 99% oligodendrocytes.
Therefore, the present invention includes oligodendrocyte-enriched cultures
comprising at least about 20%, preferably at least about 30%, more preferably
at least
about 40%, yet more preferably at least about 50%, and even more preferably at
least
about 60%, 70%, 80% or 90%, oligodendrocytes in the population.
Oligodendrocyte-enriched cultures derived from ES cells are
exemplified in the present invention. However, other pluripotent cell types
can be
differentiated into neural cells; therefore, oligodendrocyte-enriched cultures
derived
from any pleuripotent cell type are included in the invention. These include,
but are
not limited to, progenitor cells from the developing nervous system, cells
derived
from naturally occuring carcinomas and embryonal carcinoma cells, such as P19
(Bain
et al., 1998), among others.
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Since basic features of pleuripotent cells are consistent across species,
oligodendrocyte-enriched cell cultures from such cells of any vertebrate are
included
in the invention. In a preferred embodiment, the oligodendrocytes are produced
from
mammalian cells; particularly preferred are primate cells, and most preferred
are
human cells. Other vertebrate cells from which oligodendrocyte-enriched
cultures can
be obtained include, but are not limited to, laboratory rodents such as mice
(exemplified herein), rats (also exemplified herein) and hamsters, and
domesticated
animals such as cats, dogs and horses.
Presented with the present invention is a method to make a highly
purified culture of oligodendrocytes. This method comprises the several steps
of:
1. typsinizing and triturating mixed cultures of oligodendrocytes, neurons,
and
astrocytes from 4-/4+ stage embryoid bodies (to dissociate the cells);
2. culturing the cells in culture flasks with modified SATO medium (as
described
below) for a specified period (e.g., 3 days in vitro (DIV));
3. gently shaking the flasks to suspend loosely adhering cells (primarily
oligodendrocytes) while astrocytes remain adhering to the flasks; and
4. transfernng the suspended cells to a new flask containing modified SATO
medium
at a 1:1 ratio for an additional culture period.
Example 3 sets forth a detailed set of instructions on how to prepare an
oligodendrocyte-enriched culture from ES cells.
This last step, in which the cells are further cultured in a medium that
contains both fresh SATO medium and "old" medium that is oligsphere-
conditioned,is key to culturing this highly enriched culture. While not
limiting the
operation of this culture method to any one explanation, it is likely that
differentiated
oligodendrocytes condition the medium in which they grow with a factors) that
selectively promotes the survival and proliferation of oligodendrocytes. The
culture
method produces oligospheres, which are primarily composed of immature and
mature oligodendrocytes and nestin positive progenitor cells.
This novel oligodendrocyte culture method has several advantages over
previous methods. The few astrocytes that are generated in this method adhere
to the
side of the culture flask and are easily removed from the culture.
Additionally, the
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free-floating spherical cell clusters (oligospheres) can be easily
concentrated or moved
to a different medium for use.
The method to produce an enriched oligodendrocyte culture may be
used with EB cells from other species. Examples 1 and 2 teach the use of the
method
with Mus musculus and Rattus EB cells, illustrating the flexibility of the
method. Due
to the great similarity between EB cells from all mammalian species, it is
contemplated that the culture method may be used with equal efficacy with EB
cells
of other species. Species of particular interest include, but are not limited
to, human
and monkey.
Bioengineered stem cell cultures may also be used to advantage with
the culture method. Autologous ES cells are particularly advantageous when the
cells
are to be used for transplantation. Autologous ES cells may be created by
transferring
a ES cell nucleus into a denucleated cell from the patient. These autologous
ES cells
will have the totipotent characteristics of the ES cell but will not be
rejected when
transplanted into the patient. Embryonic stem cell cultures from the same
species
may also be genetically engineered to remove markers that will cause rejection
in the
patient.
Also presented with the invention is a method to regenerate the central
nervous system in patients that are deficient in axon myelination, which
encompasses
introducing oligodendrocyte cells cultured in vitro from embryonic stem cells.
The
use of in vitro cultures is superior to previous methods of transplanting in
vivo
differentiated cells because it is known that the ES cells are genetically
normal while
in vivo differentiated populations may be harboring tumor cells. The use of ES-
derived oligodendrocytes is also less invasive than previous methods where
precursor
cerebellar cells were used.
Embryonic stem cells are also advantageous over in vitro differentiated
cells because they can be easily genetically modified. The ES cells may be
engineered
to produce factors that will aid spinal cord regeneration and/or make the cell
more
likely to survive transplantation, i.e. tolerance of low oxygen conditions.
Factors of
particular interest for spinal cord reneration include, but are not limited
to,
neurotrophin 3 (NT-3), ~3-FGF and L1. The use of embryonic stem cells to
culture an
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oligodendrocyte culture is particularly advantageous because it allows cells
that are
genetically identical to the patient to be treated.
While the use of enriched cultures of oligodendrocytes for
transplantation therapy is discussed above, this enriched cell culture is also
extremely
valuable for research purposes. The oligodendrocyte culture of the invention
may be
used to study disorders of oligodendrocyte cells found in humans, the
mechanisms
that regulate production of oligodendrocytes and the factors that the cells
produce to
regulate the development of other oligodendrocytes. This culture may also be
used to
develop an in vitro model system of neural tissue that will be invaluable for
understanding how such tissues work and can be regenerated.
The following examples are provided to describe the invention in
greater detail. They are intended to illustrate, not to limit, the invention.
EXAMPLE 1
Preparation and Use of In Vitro
Differentiated Neural Cells to
Regenerate the Central Nervous System
METHODS
Cell culture. D3 or ROSA26 mouse ES cells were maintained and
differentiated in culture according to the 4-/4+ protocol of Bain et al. (Dev.
Biol. 168,
342-357 (1995)). Undifferentiated ES cells were propagated in the presence of
leukemia inhibitory factor (LIF, Gibco). Cells were cultured as embryoid
bodies in
the absence of LIF for 4 days, then treated for 4 days with retinoic acid (all-
tra~zs-RA,
500 nM, Sigma). On the 9''' day, embryoid bodies were partially trypsinized (S
min. at
37°C, 0.25% trypsin with EDTA) and resuspended in ES cell media (Bain,
et al., Dev.
Biol. 168, 342-357 (1995)) prior to transplantation.
Spinal cord injury. Impact injury was induced using the weight drop
device developed at New York University as described previously (Basso, et
al., J.
Neurotrauma 12, 1-21 (1995); Liu, et al., J. Neurosci. 17, 5395-5406 (1997)).
Adult
Long Evans female rats (275 X25 g; Simonsen Lab, Gilroy, CA) were anesthetized
with pentobarbital (50 mg/kg, i.p.), a laminectomy performed at T9-10 level,
and the
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dorsal surface of the cord subjected to weight drop impact, using a 10 gm
weight (2.5
mm diameter) dropped at a height of 25 mm (Liu, et al., J. Neurosci. 17, 5395-
5406
(1997)). During surgery, the rectal temperature was maintained at 37.0 ~ 0.5
°C by a
thermostatically-regulated heating pad (Versa -Therm 2156, Cole-Parmer,
Chicago,
IL), and during recovery, rats were placed in a temperature- and humidity-
controlled
chamber (Thermocare Inc., Incline Village, NV) overnight.
Transplantation. BBB scores were obtained the day before
transplantation (day 8 post-injury) and control and experimental groups were
matched
and randomly assigned to ensure that initial locomotor scores were equalized
between
groups. The weight-drop injury level was chosen based on previous experience
with
the NYLT impact model, to produce spontaneous recovery at a BBB score 8, the
most
sensitive portion of the scale corresponding to absent weight supported
walking. Nine
days after impact injury, rats received transplants of neural differentiated
ES cells
(approximately 1 million), vehicle medium, or 1 million adult mouse
neocortical cells
using a spinal stereotaxic frame, a 100 ~m diameter tip glass pipette
configured to a 5
~.1 Hamilton syringe, and a Kopf microstereotaxic injection system (Kopf Model
5000
& 900). Five ~1 of the ES cell or mouse neocortical cell suspension or vehicle
medium was injected into the center of the syrinx at the T9 level over a 5
minute
period. Three independent experiments with time matched controls were
completed
in total. The first series was completed for behavioral analysis and late
histologic
analysis [5 weeks post-transplantation, n = 11 per group, ES cell
transplantation vs.
vehicle medium control, D3 ES line used]. The second series was used to
compare
early (2 weeks post-transplantation) and late (5 weeks post-transplantation)
histological outcomes [n = 11 per group, ES cell transplantation (ROSA lac-Z
transgene line) vs. vehicle medium control]. In the third series, 3 groups
were
compared for behavioral outcome to assess the effects of rat immune reactions
to
mouse cells [n = 6 per group, neural differentiated ES cell transplantation
(ROSA26
lac-Z transgene line) vs. mouse neocortical cell transplantation vs. vehicle
medium
control, survival to 5 weeks post transplantation]. All groups received the
same
cyclosporine immunosuppression.
Animal care. All surgical interventions and animal care were
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provided in accordance with the Laboratory Animal Welfare Act, the Guide for
the
Care and Use of Laboratory Animals (NIFI, EHEW Pub. No. 78-23, Revised, 1978)
and the Guidelines and Policies for Rodent Survival Surgery provided by the
Animal
Studies Committee of Washington University School of Medicine. Manual bladder
expression was performed 3 times daily until reflex bladder emptying was
established.
Cyclosporine (10 mg/kg, s.q.) was administered daily to all animals in every
group
beginning the day of transplantation.
Behavioral testing. Behavioral testing was performed weekly by two
individuals blinded to treatment using the BBB Locomotor Rating Scale (Basso,
et al.,
J. Neurotrauma 12,1-21 (1995)). Behavioral outcomes and examples of specific
BBB
locomotor scores were recorded using digital video.
Immunohistochemistry. Primary antibodies used were directed
against the following antigens: astrocytes, GFAP rabbit polyclonal, 1:4
(Incstar);
oligodendrocytes, APC CC-1 mIgGl, 1:400 (Calbiochem Oncogene Sciences);
neurons, NeuN mIgG,, 1:500 (Chemicon); anti-mouse EMA rat hybridoma, 1:1
(Baumrind, et al., Dev. Dyn. 194, 311-325 (1992)); anti-mouse M2 rat hybridoma
(Lagenaur & Schachner J. Supramol. Struct. Cell Biochem. 15, 335-346 (1981));
anti-
BrdU mIgGl or rat polyclonal, 1:400 (Boehringer-Mannheim); anti-(3-
galactosidase
mIgG, 1:5,000 (Promega). Species-specific secondary antibodies (1:200) were
conjugated to Cy3, FITC (Jackson Immunoresearch) or Alexa 488 (1:200,
Molecular
Probes) and sections were counterstained with Hoechst 33342. Control slides
with
primary or secondary antibodies omitted were performed with each series.
Cell Quantification. Surviving BrdU positive ES cells and those
double labeled for markers of differentiated neural cells were counted in 3
longitudinal sections, centered at the middle of the cord and separated by 200
~m and
averaged per animal.
RESULTS
Neural differentiated mouse embryonic stem (ES) cells were
transplanted into a rat spinal cord 9 days after traumatic injury.
Histological analysis
2-5 weeks later revealed that transplant-derived cells survived and
differentiated into
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astrocytes, oligodendrocytes and neurons, and migrated up to 8 mm away from
the
lesion edge. Furthermore, gait analysis revealed that transplanted rats
exhibited
hindlimb weight support and partial hindlimb coordination not found in sham-
operated controls or controls transplanted with adult mouse neocortical cells.
In the first two series of studies, thoracic spinal cord injury was
induced by weight-drop (a 10 gm rod, 2.5 mm in diameter, falling 25 mm) in 22
adult
female Long-Evans rats (Basso, et al., J. Neurotrauma 12,1-21 (1995); Liu, et
al., J.
Neurosci. 17, 5395-5406 (1997)). Nine days later, 4-/4+ stage ES cell embryoid
bodies derived from the D3 line (Bain, et al., Dev. Biol. 168, 342-357 (1995))
were
partially trypsinized (5 min. at 37°C, 0.25% trypsin with EDTA) and
cell aggregates
(total of one million cells in 5 ~,1 ES cell medium) were transplanted into
the syrinx
formed at the initial site of spinal cord contusion (n=11). Sham-operated
control
animals were handled identically, including treatment with cyclosporine, but
in place
of cell transplantation they received intra-syrinx inj ections of culture
medium alone
(n=11). Beginning on the day of transplantation, all rats received
cyclosporine (10
~.g/kg s.q.) daily to prevent rejection. Hindlimb motor function was assessed
using
the Basso-Beattie-Bresnahan (BBB) Locomotor Rating Scale (Basso, et al., J.
Neurotrauma 12,1-21 (1995)). In another series of 11 animals (plus 11 sham-
operated
controls), rats received the same transplantation procedure but using ROSA26
ES
cells [a lacZ transgene containing, (3-galactosidase ((3-gal)-expressing mouse
ES cell
line] and animals were sacrificed 2 weeks after transplantation for histology
and
quantitative cell counting.
Mouse ES cell-derived cells marked genetically (using the ROSA26 (3-
gal-expressing line) and pre-labeled in vitro with BrdU (24 hr pulse, 10 ~,M)
could be
identified in situ 14-33 d after transplantation; alternatively identification
could be
achieved with the mouse specific antibodies M2 (Lagenaur & Schachner J.
Supramol.
Struct. Cell Biochem. 15, 335-346 (1981)), EMA (Baumrind, et al., Dev. Dyn.
194,
311-325 (1992)) or Thy 1.1/1.2 (data not shown for EMA and Thy 1.1/1.2). When
examined 2-5 weeks after transplantation, ES cell-derived cells were found in
aggregates or dispersed singly throughout the injury site; furthermore single
cells
could be found as far as 8 mm away from the syrinx edge in either the rostral
or
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caudal direction (Fig. 1). In the majority of the transplanted rats, by 2
weeks after
transplantation ES cell-derived cells filled the space normally occupied by a
syrinx in
medium-treated rats. By 5 weeks, the density of ES cell-derived cells in this
area was
reduced and replaced with an extracellular matrix containing fibers positive
for Thy
1.1/1.2 labeling. The other mouse-specific markers, M2 and EMA, offered
advantages over the genetic and DNA markers (which only mark cell bodies) in
that
they also labeled ES cell-derived processes, which were abundant in ES cell
transplanted rats, but not present in sham injected rats.
Surviving ES cell-derived cells, labeled with antibodies against mouse-
specific markers or BrdU, also labeled with antibodies against markers
specific for
oligodendrocytes (adenomatous polyposis coli gene product, APC CC-1),
astrocytes
(glial fibrillary acidic protein, GFAP), and neurons (neuron-specific nuclear
protein,
NeuN); nuclei could be clearly identified with Hoechst 33342 staining. Most
surviving ES cell-derived cells were oligodendrocytes (43 ~6% of BrdU labeled
cells
were O1 labeled, n = 11 rats, mean ~SEM) and astrocytes (19 ~4% were GFAP
labeled), but some ES cell-derived neurons (8 ~5% were NeuN labeled) were also
found in the middle of the cord. Many of the ES cell-derived oligodendrocytes
were
also immunoreactive for myelin-basic protein, an integral component of myelin.
No
evidence of tumor formation was observed.
Performance in open field locomotion was enhanced by ES cell
transplantation (Fig. 2). In contrast to the inability of the sham
transplantation group
to support weight with their hindlimbs, the ES cell transplant group
demonstrated
partial weight-supported ambulation. A statistical difference in BBB scores
was
achieved by two weeks following transplantation (Fig. 2a). After one month, a
difference of two points on the BBB scale was observed between groups: 7.9
X0.6 for
sham vehicle transplantation group, 10.0 X0.4 for ES cell transplant group.
The
former score signifies a gait characterized by no hindlimb weight bearing and
no
coordinated hindlimb movements, whereas the latter score signifies a gait
characterized by partial hindlimb weight bearing and partial hindlimb
coordination.
A third experimental series examined the possibility that a rat versus
mouse immune response could contribute to the observed behavioral benefit. The
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transplantation of 4-/4+ ES cells (ROSA26 line) 9 days after injury was
compared
directly to two control groups - culture medium injection and transplantation
of adult
mouse neocortical cells (n = 6 per group). Immuno-histologic examination of
the
spinal cords 5 weeks after transplantation, using antibodies directed against
microglia
/ macrophages (CDllb) and INF-'y, revealed that a similar degree of
inflammation was
present in all 3 groups. Improved locomotor function as assessed with the BBB
locomotor scale (with assignments made using slow motion video) was again
associated only with ES cell transplantation (Fig. 2b).
In summary, it has been demonstrated that mouse ES cell-derived cells,
when transplanted into the spinal cord 9 days after weight drop injury: (1)
survive for
at least 5 weeks; (2) migrate at least 8 mm away from the site of
transplantation; (3)
differentiate into astrocytes, oligodendrocytes, neurons without forming
tumors; and
(4) produce improved locomotor function. Behavioral recovery similar in
magnitude
to that shown here has previously only been shown in acute injury models
(Bernstein,
et al., Exp. Neurol. 98, 633-644 (1987); Bregman, et al., Exp. Neurol. 123, 3-
16
(1993); Howland, et al., Exp. Neurol. 135, 123-145 (1995); Grill; et al., J.
Neurosci.
17: 5560-5572 (1997)). Factors possibly responsible for the benefits observed
include
enhancement of myelination, reduction of delayed oligodendrocyte death, or
enhancement of host axonal regeneration, for example by providing a favorable
substrate for regrowth, or by producing growth factors.
EXAMPLE 2
Method to Make a Highly Enriched
Oligodendrocyte Culture
A method for producing enriched cultures of ES cell-derived
oligodendrocytes has been developed and the resulting oligodendrocytes are
capable
of myelinating axons in vitro and in vivo after transplantation in the injured
and
dysmyelinated spinal cord. In the first model, localized chemical
demyelination
injury, without damaging passing axons, was induced in the dorsal column white
matter of rats. The second model utilized myelin-deficient shiverer (shilshi)
mutant
mice that lack myelin basic protein (MBP).
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MATERIALS AND METHODS
Animals and Care. Homozygous (shilshi) shiverer mice and female
Long-Evans rats were obtained from Jackson (Bar Harbor, ME) and Simonsen
(Gilroy, CA) labs, respectively. Interventions were in accordance with the
Laboratory
Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals
(NIFI,
EHEW Pub. No. 78-23, Revised, 1978) and the Guidelines and Policies for Rodent
Survival Surgery provided by the Animal Studies Committee of Washington
University School of Medicine. Anesthesia was induced by ketamine/medetomidine
(75:0.5 mg/kg for rats, 75:1 mg/kg for mice; i.p.) and reversed with
atipamezole (1.0
mg/kg, s.q.). All animals received 1) cyclosporine (10 mg/kg, s.q:) 24 hrs
prior to
transplant and daily thereafter, 2) antibiotics (enrofloxacin, 2.Smglkg, s.q.)
prior to
surgery and daily for 3-5 days, 3) saline (2-10 ml i.p.) post-surgery and for
2 days, and
4) nutritional supplements for 3-5 days after surgery.
ES Cell Cultures. D3 (lacZ-) or ROSA26 (lacZ+) mouse ES cells
were differentiated using the 4-/4+ protocol (Bain, et al., (1995) Dev. Biol.
168, 342-
357; Example 1). After 8 days in vitro, the 4-/4+ stage floating embryoid
bodies
(EBs), were partially trypsinized (5 min. at 37°C, 0.25% trypsin with
EDTA) and
resuspended in ES cell media (ESIM) (Bain, et al., (1995) Dev. Biol. 168, 342-
357;
Example 1) for transplantation or further triturated to a single cell
suspension for
culturing in ESIM (Bain, et al., (1995) Dev. Biol. 168, 342-357; Example 1) or
modified SATO defined media with or without serum (Bottenstein & Sato, (1979)
Proc. Natl. Acad. Sci. USA 76, 514-7; Raff, et al., (1983) Nature 303, 390-
396).
Demyelination. Demyelination of dorsal column white matter was
induced chemically in rats using characterized methods (Hall, (1972) J. Cell
Sci. 10,
535-546; Blakemore, (1976) Neuropathol. Appl. Neurobiol. 2, 21-39; Waxman, et
al.,
(1979) J. Neurol. Sci. 44, 45-53; Blakemore, & Crang, (1985) J. Neurol. Sci.
70, 207-
223). After a T10 laminectomy, ethidium bromide (1 ~l of 0.1 % ethidium
bromide
in 0.9% saline) or lysophosphatidyl choline (LPC - lysolecithin; 2 ~,1 of 1.0%
LPC in
0.9% saline) was injected at a depth of 0.5 mm in the dorsal column over a 10
min
period using a stereotaxic microinjector (Stoelting) and a 30 ~m tip glass
pipet
attached to a 5 ~1 Hamilton syringe. Three days later, the demyelinated areas
were
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transplanted with partially dissociated EBs.
Preparation of Cells for Transplantation. Pre-labeled (see Cell
Tracking methods below) 4-/4+ stage EBs or oligospheres were prepared for
transplantation using methods described previously (Example 1) to produce
suspensions of small clusters of cells. Cell density was calculated using a
hemocytometer and adjusted to 50,000 viable cells per ~,1.
Transplantation. Demyelination injury rats received transplants of
approximately 125,000 cells from partially dissociated 4-/4+ EBs or media
vehicle. A
50-100 ~,m tip diameter glass pipet was stereotaxically advanced 0.5 mm into
the
dorsal column white matter. Using a stereotaxic microinjector, 2.5 ~1 of the
ES cell
suspension or vehicle media was injected at a rate of 0.25 ~,1/min. The needle
was left
in place for S more min., slowly withdrawn, and the laminectomy site was
covered
with artificial dura. In the second model, shiverer (shilshi) mice were
transplanted
with 100,000 oligosphere cells or vehicle medium (n > 6 each) at the T8 & T10
level
(1 ~.l at 2 sites 0.35 ~m below the dura).
Cell tracking and immunohistochemistry. Several methods were
used to track ES cells after transplantation: (1) lacZ transgene, (2) bromo-
deoxyuridine (BrdU) DNA labeling, (3) fluorescent cell tracker orange, and/or
(4)
mouse specific antibodies. ROSA26 ES cells stably expressing lacZ were used in
all
transplantation experiments (Friedman,& Soriano, (1991) Genes Dev. 5, 1513-
1523).
ES cells were pulse labeled with BrdU (10 ~,M; Boehringer-Mannheim,
Indianapolis,
IN) for 24 hours on the 3rd to 4''' day of the 4-/4+ protocol (Example 1 ). In
addition,
partially trypsin-dissociated 4-/4+ EBs were incubated with stable fluorescent
marker
cell tracker orange (Molecular Probes, Eugene, OR) for 20 min, washed,
incubated for
another 20 min, and then washed prior to transplantation. Cell tracker orange
diffuses
into cells and is transformed into a fixable, membrane-impermeant form in the
cytoplasm.
Mouse-specific antibodies were used to detect the mouse ES cells in
the rat demyelination experiments: anti-M2 (labels mouse glia > neurons), and
anti-
EMA (labels mouse neurons > glia). Antibodies used to identify the
oligodendrocyte
lineage included: anti-NG2 for oligodendrocyte progenitors (Chemicon), anti-04
for
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immature oligodendrocytes (hybridoma), anti-O1 for mature oligodendrocytes
(hybridoma), anti-MBP for terminally mature oligodendrocytes (Boehringer-
Mannheim). Homozygous shiverer (shilshi) mice are devoid of MBP and the
presence of MBP(+) myelin in these animals following transplantation provided
a
useful marker for identification of transplanted oligodendrocytes.
Electron Microscopy. EBs and cultures were processed using
standard methods (Mulvey, et al., (1998) Science 282, 1494-1497). Samples were
viewed with a Hitachi S-450 Scanning Electron Microscope operated at 20 KV
accelerating voltage and JEOL 100CX Transmission Electron Microscope.
RESULTS
ES cells differentiated using the 4-/4+ retinoic acid protocol (Bain, et
al., (1995) Dev. Biol. 168, 342-357) produce oligodendrocytes when
transplanted into
injured spinal cord (Example 1). Based on this protocol, methods were
developed for
reliable generation of mixed cultures of oligodendrocytes, neurons and
astrocytes
from 4-/4+ stage EBs. EBs floated in cell clusters, many of which contained
internal
cysts. Ultrastructural SEM examination revealed that the cells on the surface
were
covered with extensive microprocesses.
Immunohistochemical studies of EBs showed limited expression of
markers of differentiated neural cells and less than half of the cells were
nestin
positive - an early marker of neural precursors. Most cells that expressed
markers of
differentiated neural cells were confined to the exterior of the EBs, although
a subset
of cells surrounding the internal cysts were also frequently labeled with
neuronal
markers. Ultrastructural evidence suggested that a substantial number of EB
cells
exhibit features of apoptotic death. Additionally, chromatin condensation
(visible in
Hoechst stained nuclei), consistent with cell death, was present in 10-20% of
cells.
35
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Table 1. Expression of differentiated cells in EB's and oligospheres.
Percentage
of EB and oligosphere cells immunoreactive for stage specific phenotypic
markers (mean ~SEM, n = 5 each).
Cell Type NeuN GFAP 04 O1 Nestin
Single EB 0.06 0.13 0.38 0.35 0.08
X0.020 X0.011 X0.038 X0.052 X0.013
Oligosphere ~ 0.27 0.06 0.50 0.54 0.09
10.044 10.01 S X0.094 10.091 10.091
Ratio I 4.50 0.46 1.32 1.54 1.13
Further culturing of dissociated 4-/4+ EBs in standard neural media
produced mixed cultures of neurons, astrocytes and oligodendrocytes. Like
primary
cultures, ES-derived type-I astrocytes formed a confluent layer adherent to
the bottom
of the dish, other cells grew on top, and neurons grew in small clumps with
large
bundles of axons radiating outward. Mixed cultures grew best in SATO defined
media supplemented with serum and could be maintained for at least one month.
Oligodendrocyte longevity was enhanced by the presence of neurons. Adding
(3FGF
(10 ng/ml) during the first week in vitro enhanced oligodendrocyte production
and
inhibition of cell division (10'5 M cytosine arabinoside), as employed in
previous
studies of cultured ES derived neurons (Bain, et al., (1995) Dev. Biol. 168,
342-357),
limited oligodendrocyte viability.
Using immunohistochemical markers as well as scanning and
transmission electron microscopy, it was observed that ES cell-derived
oligodendrocytes produced myelin. Individual oligodendrocytes that myelinated
multiple axons and multiple segments of single axons, could be easily
identified using
fluorescent antibodies directed against a component of myelin (O1) found in
mature
myelinating cells. After 9 DIV, axonal myelin profiles with 2-3 loosely
wrapped
layers were common. It is known that development of compact mature myelin
profiles in vitro typically takes 3-6 weeks, and limited survival of neurons
past 14
DIV in our mixed cultures precluded examination at later stages in the current
studies.
In the absence of axons, oligodendrocytes formed sheets of myelin, similar to
cultures
of primary oligodendrocytes.
Enriched cultures of oligodendrocytes were produced by development
of an intermediate in vitro stage termed "oligospheres". To produce
oligospheres, 4-
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/4+ EBs were trypsinized and triturated, then placed in T25 flasks containing
5 ml of
pre-conditioned "oligosphere media" consisting of SATO defined media, ~3-FGF
(10
ng/ml) and PDGF (2 ng/ml). This media promoted survival and proliferation of
oligodendrocytes. On the fourth day, non-adherent cells (primarily
oligodendrocyte
precursors) were passed into fresh oligosphere media at a 1:1 ratio. The few
astrocytes generated adhered to the flask and were not passed. Over 6-8 days,
the
cells developed as free-floating spherical cell clusters.
Immunohistochemical studies suggested that oligospheres contained a
small number of neurons, few astrocytes, a large number of immature and mature
oligodendrocytes, and substantial numbers of nestin positive progenitor cells
(Table
1). Plating dissociated oligospheres produced cultures comprised of 92 ~7%
oligodendrocytes (n = 4), the balance neurons. One-week survival periods could
be
readily attained and longer-term growth was possible if cultures were fed with
media
conditioned by oligospheres.
To evaluate differentiation of ES cells in vivo, in the "injured" adult
CNS, partially dissociated 4-/4+ EBs were transplanted into the dorsal column
of rat
spinal cord 3 days after chemical demyelination. Successful engraftment in the
demyelinated region was evident in 9/10 rats when examined 1 week after
transplantation, as indicated by immunostaining with anti-mouse specific
antibodies,
lacZ expression, and by an increased cell density demonstrated by Hoechst
33342
labeling in transplanted animals. In rats that received a sham vehicle
transplant, axons
of passage were largely spared and a paucity of Hoechst nuclear labeling was
present
at the demyelination site. At the lesion site of transplanted rats, ES cells
differentiated
primarily into oligodendrocytes (anti-APC CC-1), but not astrocytes. Enhanced
GFAP reactivity was consistently observed at the lesion borders in both ES
cell and
vehicle medium transplanted rats, indicating the association with host
reactive
astrocytes. Little evidence of ES cell-derived neurons (anti-NeuN or anti-
neuron
specific enolase) was found in the zone of demyelination or in host tissues.
Nine of
the ten rats that received transplants exhibited this pattern of ES cell
differentiation.
Histologic evidence of acute graft rejection was present in 1/10 rats.
A second study was performed to assess the potential for ES
oligodendrocytes to myelinate in the dysmyelinated adult CNS. Dissociated
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oligospheres were transplanted into the thoracic spinal cord of shiverer
(shilshi) mice
(>2 months old), which lack MBP, an essential component of functional myelin.
Transplanted cells were tracked by pre-labeling the oligospheres with the
fluorescence
marker cell tracker orange or by detecting MBP expressed by transplanted
cells, but
absent in the host shiverer (shilshi) mice. Two weeks after transplantation,
ES cell-
derived cell tracker orange(+) and MBP(+) oligodendrocytes predominated in
white
matter. ES oligodendrocytes conformed to the organization that
oligodendrocytes
normally respect in white matter: ES oligodendrocytes would align with host
intrafascicular oligodendrocytes and myelinate axons. Since homozygous
shiverer
mice do not exhibit substantial MBP immunoreactivity, MBP immunoreactivity
could
be attributed to the transplanted ES cell-derived oligodendrocytes. In
oligosphere-
transplanted mice, but not sham-transplanted mice, widespread MBP
immunoreactivity was evident in regions surrounding the sites of
transplantation and
the pattern of MBP expression was similar to that found in the normal spinal
cord of
mice. The longitudinal parallel arrays of MBP immunoreactivity separated by
spaces
occupied by axons in white matter is characteristic of axonal myelination.
The experiments described in this example demonstrate that ES cells
can be used to reliably generate mixed and enriched cultures of
oligodendrocytes and
that these oligodendrocytes are capable of producing myelin and myelinating
axons in
vitro. In addition, transplanted ES cells can: 1 ) preferentially
differentiate into
oligodendrocytes in areas of demyelination, suggesting that environmental cues
in the
injury site can direct ES cell differentiation, and 2) myelinate host axons in
the
dysmyelinated spinal cord.
This is believed to be the first demonstration of the ability of ES cell
derived oligodendrocytes to myelinate in vitro and to survive and myelinate
axons in
the mature CNS after transplantation. These findings in the mature CNS are
particularly relevant since the most common disorders that are targets for
therapeutic
strategies of remyelination are in adults. In particular, the data demonstrate
that
injured demyelinated areas of the adult CNS may preferentially stimulate
oligodendrocyte differentiation.
Remyelination is an enticing mechanism potentially underlying the
rapid recovery of locomotor function observed when dissociated 4-/4+ stage ES
cells
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were transplanted 9 days after moderate contusion injury in rats (Example 1).
Significant recovery of locomotion was first evident 11 days after
transplantation and
oligodendrocytes represented the largest differentiated population of ES cell
derived
cells in that study.
The above results also suggest that local conditions in the lesioned
CNS can select for differentiation or survival of particular types of ES-
derived neural
cells. When ES cells are transplanted into a contusion- injured spinal cord,
they
differentiate into substantial numbers of astrocytes and oligodendrocytes
arranged in
specific patterns relative to one another (Example 1 ). In contrast, it is
shown here that
primary demyelinated lesions, sparing passing axons, preferentially induce ES
cells to
differentiate into oligodendrocytes. This observation is compatible with the
previous
demonstration that CNS isolated progenitors differentiate into different
neuronal
phenotypes based on their site of implantation in the CNS (Vicario-Abejon, et
al.,
(1995) J. Neurosci. 15, 6351-6363; Brustle, et al., (1995) Neuron 15, 1275-
1285;
Shuhonen, et al., (1996) Nature 383, 624-627). No previous report has
suggested that
CNS context can select for differentiation of oligodendrocytes from neural
progenitors.
No evidence of ES cell derived tumor formation was found in any of
the above-described in vivo studies or in our previous spinal cord contusion
transplantation series (Vicario-Abejon, et al., (1995) J. Neurosci. 15, 6351-
6363).
Formation of teratomas or other tumor types remains a concern in any
transplantation
study. A heartening feature of ES cells is that they are the only stem cells
that can be
proven genetically normal by generating a normal animal after implantation
into
blastocysts.
EXAMPLE 3
Instructions on Preparation of
Oli~,ospheres from ES Cells
This example sets forth detailed instructions on the preparation of an
oligodendrocyte-enriched cell culture from ES cells. To the extent specific
media,
reagents and materials are mentioned, they are intended to be illustrative,
not limiting,
of the invention.
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The process of culturing oligospheres is outlined in this example from
the point where Embryoid Bodies (EBs) have been successfully created (Example
1;
Bain et al, 1998). There are many stock solutions used as components of this
recipe
that must be created prior to beginning a particular step. Some of these must
be made
fresh, others can be stored and used many times. It is advisable to read
through the
entire protocol first, to be sure that all materials and stock solutions have
been
collected and/or made up prior to beginning any recipe. Each recipe outlines
the
process of making a particular medium or constituent that will be used in the
process
of making oligospheres. Further background information may be obtained from
Bain
et al.'s 1998 review, "Neuron-like Cells Derived in Culture From P19 Embryonal
Carcinoma and Embryonic Stem Cells." This review can be found online at
http://thalamus.wustl.edu/~ottlieblab/ ot~~ tlieb lab 3.html.
SATO 100X Stock Recipe
Procedural Notes: SATO 100X stock solution is the medium supplement basis for
SATO-no-serum medium and is the first series of steps in the process of making
oligospheres. All solutes can be weighed outside the hood since the SATO
100X stock will be sterile filtered before it is aliquoted and frozen down. In
the
interest of time efficiency, weigh out all solutes at once, make up individual
stock solutions, then combine them and filter. It is also advisable to pre-
label
each weighing boat and glass culture tube with the substance it will contain
to
assure that mistakes are not made.
1) Obtain all constituent solutes and, outside the hood, weigh them in
separate
pre-labeled weighing boats.
a) Crystalline BSA 200 mg
b) Progesterone 5 mg
c) Putrescine 32 mg
d) L-thyroxine 2.4 mg
e) Tri-idothyronine 2.4 mg
f) apo-Transferrin 180 mg
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g) Sodium Selenite 4 mg
2) Stock Progesterone Solution: dissolve 5 mg progesterone powder into 200
~.L sterile filtered 100% ethanol in a 5 mL roundbottom tube. If you have
already made filtered 100% ethanol, skip to step e.
a) Obtain a 60 cc sterile syringe tube and remove the plunger.
b) Pour ~50 mL 100% ethanol into syringe tube and attach a 25 mm
sterile 0.2 ~,m filter.
c) Push ethanol through filter into a sterile 50 mL conical tube.
d) Label tube with "Filtered 100% EtOH", the date, and your initials.
(This filtered 100% EtOH can be used repeatedly if sterile technique is
maintained.)
e) Carefully pour 5 mg progesterone powder into a 5 mL roundbottom
tube.
f) Using a micropipette, add 200 ~L sterile filtered 100% EtOH to the
progesterone.
g) Cap the tube and vortex until all solute is dissolved (~2 minutes).
3) Stock Thyroxine Solution: dissolve 2.4 mg thyroxine powder into 300 ~,L
sterile filtered 0.1 N NaOH in a 2°d 5 mL roundbottom tube. If sterile
filtered
0.1 N NaOH, has already been made, skip to step ~
a) Obtain a 50 mL vial of 0.1 N endotoxin-free NaOH (Sigma # 210-5).
b) Obtain a 60 cc sterile syringe tube and remove the plunger.
c) Pour 50 mL 0.1 N NaOH into syringe tube and attach a 25 mm sterile
0.2 ~.m filter.
d) Push NaOH through filter into a sterile 50 mL conical tube.
e) Label tube with "Filtered 0.1 N NaOH" and the date. (This filtered 0.1
N NaOH can be used repeatedly if sterile technique is maintained.)
f) Carefully pour 2.4 mg thyroxine powder into a 5 mL roundbottom
tube.
g) Using a micropipette, add 300 ~L sterile filtered 0.1 N NaOH to the
thyroxine.
h) Cap the tube and vortex until all solute is dissolved (~2 minutes).
4) Stock Tri-idothyronine Solution: dissolve 2.4 mg tri-idothyronine into 300
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~,L sterile filtered 0.1 N NaOH in a 3rd 5 mL roundbottom tube.
a) Repeat procedure from step 3, substituting tri-idothryonine for
thyroxine.
5) Stock Sodium Selenite Solution: dissolve 4 mg sodium selenite into 100 uL
sterile filtered 0.1 N NaOH, in a 15 mL conical tube, then add 10 mL DMEM.
a) Repeat procedure from steps 3 and 4, this time using 4 mg sodium
selenite, 100 uL sterile filtered 0.1 N NaOH, and a 15 mL sterile
conical tube.
b) Using a 10 mL serological pipette, add 10 mL DMEM to 15 mL
conical tube containing sodium selenite solution.
c) Using the same pipette, triterate solution 3X to mix.
6) Stock ITS Solution: using a 5 mL serological pipette, add 2.5 mL DMEM to
the rubber-capped commercial stock tube that the ITS came in, cap tube, and
vortex until all solute is dissolved.
7) Carefully pour each of the three remaining pre-weighed constituent powders,
(BSA, putrecine, apo-transferrin) into separate 50
mL sterile conical tubes.
8) Using a 10 mL serological pipette, place 6 mL DMEM
into each of the three
50 mL sterile conical tubes.
9) Vortex each of the three tubes until all solute is
dissolved.
10) Carefully pour the contents of each of these three
50 mL conical tubes into
one 50 mL conical tube, cap and mix by hand.
11) Using separate micropipette tips, add the designated
amount of each
individual stock solution to the 50 mL conical tube.
a) Progesterone stock solution S uL
b) L-thyroxine stock solution 10 uL
c) Tri-idothyronine stock solution 10 uL
d) ITS stock solution 2 mL
e) Sodium Selenite stock solution 150 uL
6) Using a 25 mL serological pipette, gently triterate
at least 3X to mix solution.
7) Obtain a 60 cc sterile syringe tube and remove the
plunger
8) Pour the entire solution into the syringe tube, replace
the plunger, and attach a
25 mm sterile Acrodisc 0.2 ~,m filter.
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9) Push fluid through filter into a sterile 50 mL conical tube.
10) Using a micropipette, aliquot 400 ~,L of this solution into 2.0 mL
sterile,
RNAse-free Biostore vials with caps.
11) Label vials with "SATO 100X Stock" and the date, and store at -
20°C.
S 12) Frozen SATO 100X Stock aliquots will keep for 6 months.
SATO Constituent Stocks Recipe
Procedural Notes: SATO Constituent stock solutions are the growth factor
additives
for SATO-no-serum medium and, combined, are the second series of steps in the
process of making oligospheres. All solutes can be weighed outside the hood
since the constituent stocks will be sterile filtered before they are
aliquoted and
frozen down.
In this series of steps, the practitioner will make up N-acetyl, NT-3, CNTF,
1 S Hepes, and L-glutamine stock solutions. Some stocks will keep for long
periods
of time, some will have to be made up fresh. Unless otherwise indicated,
perform these procedures in a sterile hood. In the interest of time
efficiency,
weigh out all solutes at once, make up individual stock solutions, then
aliquot
them and filter. It is also advisable to pre-label each weighing boat and
glass
culture tube with the substance it will contain to assure that mistakes are
not
made.
Sterile Filtered dd HZO and 0.01 M PBS pH7.4
13) Small aloquots of sterile filtered dd Hz0 will be needed during the
following
procedures. To make these aliquots, obtain one full Kimax 1L bottle of
sterile (autoclaved) dd HZO and place it in the hood.
14) Under the hood, attach a sterile bottletop filter and vacuum tube to an
empty
sterile Kimax 1 L bottle.
15) Pour dd HZO through filter, into bottle.
16) Using a 25 mL serological pipette, aloquot the entire volume of sterile
filtered
water into sterile SO mL tubes.
17) Tighten caps, label tubes with "Filtered dd HZO" and the date. Store
aliquots
at room temperature - these will keep. indefinitely and can be used repeatedly
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if sterile technique is maintained.
18) Obtain 50 mL of 0.01 M PBS pH 7.4 in a sterile 50 mL conical tube, pour
entire volume into a 60 cc syringe tube, attach a sterile 25 mm Acrodisc 0.2
~,m filter, and push fluid through filter and into a sterile 50 mL conical
tube.
S 19) Tighten cap, label tube with "Sterile PBS" and the date. Store at room
temperature - this solution will keep for a long time and the same aliquot can
be used repeatedly if proper sterile technique is maintained.
20) Obtain two 50 mL aliquots of sterile water and place in the hood.
21) Using a 1 mL serological pipette, take 1 mL of sterile water from one of
the
aliquots and place it in a 5 mL roundbottom tube.
22) Weigh 6.3 mg N-acetyl-cysteine outside of the hood.
23) Carefully pour powder into the 1 mL volume of sterile water in the 5 mL
Falcon tube from step 7.
24) Vortex until all powder is dissolved (this will take about 2 minutes).
25) In the hood, and using a 3 cc syringe tube, suck up NAC solution, attach a
sterile 13 mm Acrodisc 0.2 ~,m filter (Sterile Acrodisc 13; 0.2 ~,m; single
use; low protein binding; non-pyrogenic filter - Gelman Sciences # 4454) and
push fluid through filter and into another sterile 5 mL Falcon tube.
26) Label tube "NAC stock" and leave in hood - this solution does not keep and
will have to be made up fresh each time it is used.
27) 100X NAC stock is now ready
28) Obtain a 50 mL aloquot of sterile 0.01 M PBS and place it in the hood.
29) Using a 10 mL serological pipette, transfer 10 mL sterile 0.01 M PBS into
a
sterile 15 mL centrifuge tube.
30) Cap the tube and bring it to the scale.
31) Weigh 0.1 g BSA and carefully pour into the 15 mL tube from the last step
32) Cap tube tightly and vortex for 30 seconds
33) Obtain a sterile 10 cc syringe tube, attach a sterile 13 mm Acrodisc 0.2
~,m
filter, remove the plunger, pour BSA solution into syringe body, replace
plunger, and push fluid volume through filter into another sterile 15 mL
conical tube - this completes the preparation of a 1 % BSA stock solution.
34) Under the hood, and using a 1 mL serological pipette, aliquot 1 mL 1 % BSA
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stock solution into each of ten 2 mL Biostore vials.
35) Cap the vials tightly, label with "1% BSA" and the date.
36) Store 1% BSA stock at -20°C - this solution will keep for ~6
months.
37) Obtain concentrated NT-3 stock solution (gift) and five to ten 1 mL
aliquots
of the 1% BSA in PBS stock solution (the number needed depends on how
much concentrated NT-3 stock is available).
38) Thaw 1% BSA aliquots at room temperature for 5 minutes.
39) Using a micropipette, transfer 900 ~,L of the 1% BSA stock and 100 ~,L of
the concentrated NT-3 stock into a new 2 mL Biostore vial.
40) Repeat this process until there is no more concentrated NT-3 stock left.
41) Cap vials tightly and label with "100~,g/mL NT-3" and the date.
42) Store diluted NT-3 stock at -20°C - this stock will keep for ~6
months.
43) Obtain a 10 ~,g vial of CNTF powder (it comes in these quantities directly
from Sigma) and a 1 mL aliquot of the 1% BSA stock.
44) Let the 1% BSA stock thaw at room temperature for 5 minutes.
45) Under the hood, using a micropipette, add 400 ~,L of 1 % BSA stock to the
10
~g vial of CNTF (it comes pre-measured from the company).
46) Vortex solution for ~1 minute or until all powder is dissolved.
47) CNTF stock is now ready. This stock is usually made fresh each time we
need it, but many aliquots can be made up at one time and stored at -
20°C for
several months.
48) Obtain a 50 mL aliquot of sterile ddI water.
49) Weigh out 11.915 g Hepes powder and carefully pour into an empty, sterile
50 mL conical tube.
SO) Pour ddI water from aliquot into Hepes tube up to 50 mL mark.
51) Vortex until all powder is dissolved. This may take upwards of 30 minutes.
52) Fill remaining volume in tube up to 50 mL with ddI water.
53) Vortex 1 minute.
54) Obtain a sterile 60 cc syringe tube, attach a sterile 25 mm Acrodisc 0.2
~.m
filter, remove the plunger, pour Hepes solution into syringe body, replace
plunger, and push fluid volume through filter into another sterile SO mL
conical tube - this completes the preparation of Hepes stock solution.
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55) Cap tube tightly and label with "Hepes Stock"and the date.
56) Store at room temperature - this aliquot can be used repeatedly if sterile
technique is maintained and will keep on the shelf for ~4 months.
57) Obtain one 200 mM L-glutamine vial.
58) Obtain one 50 mL aliquot of sterile ddI water.
59) Under the hood, fill a 60 cc sterile syringe, with a 21 gauge needle
attached,
with the 50 mL sterile water from the last step.
60) Carefully remove aluminum cap tab and slowly inject 50 cc ddI water into
bottle.
61 ) Give the bottle two or three shakes to remove any residual powder from
top
and sides of bottle.
62) Carefully remove the aluminum and rubber stopper cap from the bottle -
this
must be accomplished without losing any powder or liquid. Save the stopper
in the sterile hood.
63) Cover the bottle opening with parafilm and incubate in a 37°C
waterbath for 5
minutes.
64) Under the hood, replace the rubber stopper cap and shake until all powder
is
dissolved - solution should appear completely clear and colorless.
65) Obtain a 60 cc sterile syringe tube, attach a sterile 25 mm Acrodisc 0.2
~,m
filter, remove the plunger, pour L-glutamine solution into syringe body,
replace plunger, and push fluid volume through filter into a sterile 50 mL
conical tube - this completes the preparation of L-glutamine stock solution.
66) Using a micropipette, aliquot 500 ~,L L-glutamine stock solution into 0.75
mL Biostore vials.
67) Tighten caps and label each vial with "200 mM L-glut" and the date.
68) Store at -20°C - this stock solution will keep for -~- 3 months and
needs to be
protected from light exposure.
69) Immediately before use, this stock must be thawed in a 37°C water
bath for 5
minutes and vortexed to redissolve any solute that has fallen out of solution
during storage.
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Embryonic Stem Cell Induction Medium (ESIM) Recipe
Procedural Notes: As with MS media, ESIM is made in 1L bottles without use of
volumetrics for exact measurement of final volumes. For this reason, it is
important
to be accurate in measuring volumes of components and consistent in the type
of
S media bottle. Again, it is important to keep sterile technique always in
mind when
making ESIM. It is advisable to weigh out all powders at once, outside the
hood, and
label each weighing boat and tube with the substance it will contain.
72) Obtain 100 mL NCS (New Calf Serum) and 100 mL FBS (Fetal Bovine
Serum).
73) Place serums in a 60°C water bath for 30 minutes to heat-inactivate
the
compliments in the serum.
74) During this water bath inactivation time, make up NS (Nucleoside Stock).
75) To make NS: weigh out all constituent nucleoside powders at one time
1 S outside the hood.
a) Adenosine 40 mg
b) Guanosine 42.5
mg
c) Citidine 36.5
mg
d) Uridine 36.5
mg
e) Thymidine 12 mg
76) Carefully pour all pre-weighed nucleoside powders into a sterile 50 mL
conical tube.
77) Pour 50 mL sterile dd HZO into the 50 mL tube containing the nucleoside
powders. This quantity of stock is enough to make 5 L ESIM and can be used
repeatedly if sterile technique is maintained.
78) Place solution into a 37°C water bath for ~15 minutes, shaking
intermittently
until solutes are completely dissolved.
79) Pour entire solution into a sterile 60 cc syringe tube, replace the
plunger, and
attach a 25 mm sterile Acrodisc 0.2 ~.m filter. Push solution through filter
into another sterile 50 mL conical tube.
80) Label with "NS" and the date. This stock will keep for 6 months and can be
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used repeatedly if sterile technique is maintained - however, before each use
of the NS, repeat step 7.
81) Obtain one of the sterilized empty Kimax 1L bottles made during the MS
recipe.
82) Obtain 1L DMEM and pour 790 mL into the empty sterile 1L Kimax bottle.
83) Pour heat-inactivated serums (NCS, FBS) directly into the 1L Kimax
containing 790 mL DMEM.
84) Using a 10 mL seriological pipette, add 10 mL NS stock to the DMEM plus
serum solution.
85) Shake bottle by hand to mix solution.
86) Attach a 500 mL bottletop filter and vacuum tube to another sterile, empty
Kimax 1 L bottle.
87) Pour ESIM solution through filter into the second bottle.
88) Cap new bottle containing medium tightly, label with "ESIM", the date,
your
initials, and store in 4°C refrigerator.
89) ESIM is now ready for use, will keep for ~30 days, and can be used
repeatedly if sterile technique is maintained.
SATO-no-serum Medium Recipe
Procedural Notes: This procedure is the final recipe for creating SATO-no-
serum
medium, the culturing medium used in creating oligospheres from EBs.
91) Obtain all constituents listed in the SATO-serum free Media Constituents
Table from their various storage locations.
92) Under the hood, using a 25 mL serological pipette, transfer 37 mL DMEM
into a sterile 50 mL conical tube.
93) Using a micropipette, transfer 400 ~.L of 100 mM MEM Sodium Pymvate
stock into DMEM solution from the last step.
94) Using a micropipette, transfer 400 ~.L of the SATO 100X stock into DMEM
solution from the last step.
95) Using a micropipette, transfer 400 ~,L of the N-acetyl-cysteine stock into
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DMEM solution from the last step.
96) Using a micropipette, transfer 20 ~.L of the NT-3 stock into DMEM solution
from the last step.
97) Using a micropipette, transfer 32 ~,L of the CNTF stock into DMEM solution
from the last step.
98) Using a micropipette, transfer 600 ~,L of the HEPES stock into DMEM
solution from the last step.
99) Using a micropipette, transfer 400 ~,L of the L-glutamine stock into DMEM
solution from the last step.
100) Using a 25 mL serological pipette, triturate 3X to mix solution.
101) Obtain a sterile 60 cc syringe, attach a sterile Acrodisc filter, remove
the plunger, pour SATO-no-serum solution into syringe body, replace
plunger, and push fluid volume through filter into another sterile 50 mL
conical tube
102) Label tube with "SATO-no-serum", the date, and your initials.
103) Store at 4°C - this medium will keep for ~ 14 days and can be used
repeatedly during this time if sterile technique is maintained.
Oligosphere Recipe
Procedural Notes: It is very important to carry out all procedures in a
sterile hood and
with sterile instruments and tubes unless otherwise indicated. Specifically,
it is
advisable to use a fresh, sterile, serological pipette for each step unless
repeated use is
indicated. Centrifugation is not generally performed in a hood, but tube caps
must be
kept tight during this procedure to insure that cells remain in a sterile
environment.
Do not allow any liquids or pipettes to contact the necks of flasks at any
time because
this part of the flask can be contaminated. To prevent any medium from
contacting
the neck when handling cells in T25 flasks, keep flasks nearly level with a
very slight
tilt toward the rear of the flask.
Finally, it is important to loosen caps on T25 flasks when cells are placed in
an
incubator. This will ensure that the atmosphere of the incubator can
equilibrate with
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the gases inside the flask. Conversely, caps should be tightened on flasks
when they
are removed from the incubator to ensure that a sterile environment is
maintained.
Gelatin-coated Flasks
1) At least 6 hours before beginning the oligosphere procedure, obtain ten
T25 flasks for gelatin coating procedure.
2) Obtain 2% sterile gelatin solution from 4°C refrigerator and let sit
under
the hood until solution is room temperature, clear, and homogenous.
3) Under the hood, using a 25 mL serological pipette, transfer 22.5 mL sterile
dd (double-deionized) H20 into a 50 mL conical tube.
4) Using a 5 mL serological pipette, add 2.5 mL 2% sterile gelatin solution.
This makes a 0.2% (1:9 dilution) gelatin solution.
5) Using a 25 mL serological pipette, triturate solution 3X to mix.
6) Using a 5 mL serological pipette, add 5 mL 0.2% gelatin solution to each
T25 flask.
7) Tighten cap and let sit at room temperature for at least 6 hours - for
optimal gelatin bonding, overnight setting is preferred. Gelatin-coated
flasks will keep at room temperature for several weeks, so these flasks can
be used for other procedures as well.
8) Remember: immediately before using any of the gelatin-coated flasks,
remove cap and tilt flask to allow liquid to run to one side. Suction off all
liquid, being careful not to scrape gelatin coated bottom.
Final SATO-no-serum Medium Preparation
9) Obtain a SATO-no-serum medium (see SATO-no-serum Medium Recipe)
aliquot from the 4°C refrigerator and let sit under the hood until
solution is
room temperature (~ 15 min.).
10) Under the hood, using a 10 mL serological pipette, transfer 10 mL SATO-
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no-serum medium to a 1 S mL conical tube.
11) Place remaining SATO-no-serum medium aliquot back in 4°C
refrigerator.
12) Using a micropipette, add 1 ~L 100~g/mL bFGF stock to the 10 mL
SATO-no-serum medium in the 15 mL conical tube.
13) Using a micropipette, add 2 ~L 10~,g/mL PDGF stock to SATO-no-serum
plus bFGF solution.
14) Using a 10 mL serological pipette, triturate 3X to mix solution (this
completes the preparation of the SATO-no-serum plus bFGF and PDGF
stock solution for steps 28 and 31 - this must be made up fresh before each
use). This quantity of stock will be enough to perform the first day's
oligosphere procedure on 1 dish of EBs.
Dissociating EBs
15) Obtain MS (Media Stock) and ESIM (Embryonic Stem Cell Induction
Medium) from the 4°C refrigerator and let them sit under the hood
until
solutions are room temperature (~ 15 min.).
16) Under the hood, using a 10 mL serological pipette, transfer EBs (4-/4+),
along with all their media, from the 100 mm Canada petri dish (this is
what the EBs are grown in) into a 15 mL conical tube.
17) Let EBs settle by gravity to bottom of tube for 5 minutes.
18) Suction off supernatant media to just above EB layer (be careful not to
suction off the EBs).
19) Using a 10 mL serological pipette, add 10 mL of MS to settled EBs (this is
used to dilute any depleted media that remains with the cells).
20) Repeat steps 17 and 18.
21) Using a 5 mL serological pipette, add 2 mL of 0.25% Trypsin containing
EDTA to the 15 mL tube.
22) Incubate for 6-8 minutes in a 5% COZ atmosphere 37°C incubator
(every
two minutes the suspension should be gently agitated by hand to aid in
dissociation).
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23) Using a 10 mL serological pipette, add 10 mL ESIM
(ES Cell Induction
Medium) to the trypsin and cell suspension (this
is used to stop trypsin
activity at a specific time point).
24) Place MS and ESIM bottles back in the 4C refrigerator.
25) Centrifuge for 5 minutes at 850 G.
26) While the centrifuge is running, suction off liquid
from two T25 gelatin-
coated flasks (careful not to scrape gel-coating
off the bottom of the flask).
See step 8.
27) Remove cells from centrifuge and, under the hood,
suction off supernatant
media to just above cell layer.
28) Using a 5 mL serological pipette, add 2 mL SATO-no-serum
medium with
bFGF and PDGF (see steps 9-14) to the cells in
the 15 mL conical tube.
29) Gently stir a micropipette tip through the cell-slurry.
Naked DNA from
dead cells should stick to the end of this pipette
tip and can be lifted
directed out of the cell slurry, then discarded.
30) Using a cotton-plugged, borosilicate glass Pasteur
pipette with a rubber
bulb attached, triturate l Ox to dissociate EBs
into single-cell suspension.
Usin g a 10 mL serological pipette, add 8 mL SATO-no-serum
medium with bFGF
and PDGF
Oligosphere Materials Table
Manufacturing Distributing
Product Name Size Companx Product No. Company Cat.
No.
Falcon Conical50 mL Becton-Dickinson2070 Fisher 14-432-22
Tube
15 mL Becton-Dickinson2095 Fisher OS-527-90
Roundbottom 5 mL Becton-Dickinson2054 Fisher 14-956-1B
Tube
Serological 50 mL Becton-Dickinson7550 Fisher 13-675-27
Pipette
25 mL Becton-Dickinson7525 Fisher 13-668-2
10 mL Becton-Dickinson7551 Fisher 13-675-20
5 mL Becton-Dickinson7543 Fisher 13-675-22
1 mL Becton-Dickinson7521 Fisher 13-675-
15B
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Biostore Vials 2 mL United LaboratoryUP 2231 ------- -------
(Sterile, RNAse Plastics
Free)
0.75 mL UP 2235 _______ ______
T25 Culture 125 mL Corning Costar3055 Fisher 07-200-72
Flask
Type B 2% Gelatin100 mL Sigma G-1393 ------- -------
0.25% Trypsin-EDTA100 mL GibcoBRL 25200-056 ------- ------
Pasteur pipette9 mL Fisher 13-678-8B ------- ------
(borosilicate
glass)
Syringe Tube 60 cc Becton-Dickinson309663 Fisher 14-823-2D
10 cc Becton-Dickinson309604 Fisher 14-823-2A
3 cc Becton-Dickinson309585 Fisher 14-829-40
Syringe Needle 21 gauge Becton-Dickinson305165 Fisher 14-826C
Bottletop Filter500 mL Nalgene 295-3320 Fisher 09-740-
37T
(PES membrane)
Acrodisc Filter25 mm Pall Gelman 4192 VWR 28144-040
(0.2 micron) 13 mm Pall Gelman 4454 VWR 28142-340
Kimax Media 1 L Kimble 61110-1000 Fisher 06-421-4
Bottle
Polypropylene 1 L Kimble 73808-33430 Fisher 03-340-30
Cap
For 1L Kimax Bottle
(Bonded PTFE liner)
The present invention is not limited to the embodiments described and
exemplified
above, but is capable of variation and modification without departure from the
scope of the appended claims.
