U.S. patent application number 15/432341 was filed with the patent office on 2017-06-08 for myelination of congenitally dysmyelinated forebrains using oligodendrocyte progenitor cells.
The applicant listed for this patent is Cornell Research Foundation, Inc.. Invention is credited to Steven A. GOLDMAN, Neeta Singh ROY, Martha WINDREM.
Application Number | 20170159015 15/432341 |
Document ID | / |
Family ID | 27757690 |
Filed Date | 2017-06-08 |
United States Patent
Application |
20170159015 |
Kind Code |
A1 |
GOLDMAN; Steven A. ; et
al. |
June 8, 2017 |
MYELINATION OF CONGENITALLY DYSMYELINATED FOREBRAINS USING
OLIGODENDROCYTE PROGENITOR CELLS
Abstract
One form of the present invention is directed to a method of
remyelinating demyelinated axons by treating the demyelinated axons
with oligodendrocyte progenitor cells under conditions which permit
remyelination of the axons. Another aspect of the present invention
relates to a method of treating a subject having a condition
mediated by a loss of myelin or a loss of oligodendrocytes by
administering to the subject oligodendrocyte progenitor cells under
conditions effective to treat the condition mediated by a loss of
myelin or a loss of oligodendrocytes. A further aspect of the
present invention relates to an in vitro method of identifying and
separating oligodendrocyte progenitor cells from a mixed population
containing other mammalian brain or spinal cord cell types.
Inventors: |
GOLDMAN; Steven A.;
(Webster, NY) ; ROY; Neeta Singh; (Pelham, NY)
; WINDREM; Martha; (West Henrietta, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Cornell Research Foundation, Inc. |
Ithaca |
NY |
US |
|
|
Family ID: |
27757690 |
Appl. No.: |
15/432341 |
Filed: |
February 14, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15161917 |
May 23, 2016 |
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15432341 |
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13527099 |
Jun 19, 2012 |
9371513 |
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15161917 |
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12111839 |
Apr 29, 2008 |
8206699 |
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13527099 |
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10368810 |
Feb 14, 2003 |
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12111839 |
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60358006 |
Feb 15, 2002 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 5/0618 20130101;
C12N 5/0622 20130101; A61P 29/00 20180101; A61P 27/02 20180101;
Y02A 50/30 20180101; C12N 5/0619 20130101; Y02A 50/467 20180101;
A61P 25/00 20180101; A61K 35/30 20130101; A61P 31/04 20180101; C12N
2510/00 20130101; G01N 33/5005 20130101; G01N 33/56966 20130101;
C12N 5/0623 20130101; A61K 35/12 20130101; A61P 43/00 20180101;
A61P 7/04 20180101 |
International
Class: |
C12N 5/0797 20060101
C12N005/0797 |
Goverment Interests
[0002] The subject matter of this invention was made with
government support under grant number NINDS R01NS39559 awarded by
National Institutes of Health. The government has certain rights in
this invention.
Claims
1. (canceled)
2. An isolated population of human cells, said population enriched
for A2B5.sup.+/PSA-NCAM.sup.-glial progenitor cells.
3. The isolated population of claim 2, wherein the human is an
adult.
4. The isolated population of claim 2, wherein the human is
post-natal.
5. The isolated population of claim 2, wherein the human is
fetal.
6. The isolated population of claim 2, wherein at least 90% of the
population comprises A2B5.sup.+/PSA-NCAM.sup.- glial progenitor
cells.
7. The isolated population of claim 2, wherein at least 95% of the
population comprises A2B5.sup.+/PSA-NCAM.sup.- glial progenitor
cells.
8. The isolated population of claim 2, wherein at least 99% of the
population comprises A2B5.sup.+/PSA-NCAM.sup.- glial progenitor
cells.
9. The isolated population of claim 2, wherein the glial progenitor
cells are oligodendrocyte progenitor cells (OPCs).
10. The isolated population of claim 2, wherein the population
contains uncommitted progenitor cells.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/161,917, filed May 23, 2016, which is a
divisional of U.S. patent application Ser. No. 13/527,099, filed
Jun. 19, 2012, now U.S. Pat. No. 9,371,513, issued on Jun. 21,
2016, which is a continuation of U.S. patent application Ser. No.
12/111,839, filed Apr. 29, 2008, now U.S. Pat. No. 8,206,699,
issued on Jun. 26, 2012, which is a continuation of U.S. patent
application Ser. No. 10/368,810, filed Feb. 14, 2003, which claims
the benefit of U.S. Provisional Patent Application Ser. No.
60/358,006, filed Feb. 15, 2002, which are hereby incorporated by
reference in their entirety.
FIELD OF THE INVENTION
[0003] The present invention is directed to the myelination of
congenitally dysmyelinated forebrains using oligodendrocyte
progenitor cells and to a method of treating a subject having a
condition mediated by a loss of myelin or a loss of
oligodendrocytes. Also disclosed is a method for the identification
and separation of oligodendrocyte progenitor cells from a mixed
population containing other mammalian brain or spinal cord cell
types.
BACKGROUND OF THE INVENTION
[0004] A broad range of diseases, from the inherited
leukodystrophies to vascular leukoencephalopathies to multiple
sclerosis, result from myelin injury or loss. In the pediatric
leukodystrophies, in particular, compact myelin either fails to
properly develop, or is injured in the setting of toxic storage
abnormalities. Recent studies have focused on the use of
transplanted oligodendrocytes or their progenitors for the
treatment of these congenital myelin diseases. Both rodent and
human-derived cell implants have been assessed in a variety of
experimental models of congenital dysmyelination. The myelinogenic
potential of implanted brain cells was first noted in the shiverer
mouse (Lachapelle et al., "Transplantation of CNS Fragments Into
the Brain of Shiverer Mutant Mice: Extensive Myelination by
Implanted Oligodendrocytes," Dev. Neurosci 6:325-334 (1983)). The
shiverer is a mutant deficient in myelin basic protein (MBP), by
virtue of a premature stop codon in the MBP gene that results in
the omission of its last 5 exons (Roach et al., "Chromosomal
Mapping of Mouse Myelin Basic Protein Gene and Structure and
Transcription of the Partially Deleted Gene in Shiverer Mutant
Mice," Cell 42:149-155 (1985)). Shiverer is an autosomal recessive
mutation, and shi/shi homozygotes fail to develop central compact
myelin. They die young, typically by 20-22 weeks of age, with
ataxia, dyscoordination, spasticity, and seizures. When fetal human
brain tissue was implanted into shiverers, evidence of both
oligodendrocytic differentiation and local myelination was noted
(Lachapelle et al., "Transplantation of Fragments of CNS Into the
Brains of Shiverer Mutant Mice: Extensive Myelination by Implanted
Oligodendrocytes," Dev. Neurosci 6:326-334 (1983); Gumpel et al.,
"Transplantation of Human Embryonic Oligodendrocytes Into Shiverer
Brain," Ann NY Acad Sci 495:71-85 (1987); and Seilhean et al.,
"Myelination by Transplanted Human and Mouse Central Nervous System
Tissue After Long-Term Cryopreservation," Acta Neuropathol 91:82-88
(1996)). However, these unfractionated implants yielded only patchy
remyelination and would have permitted the co-generation of other,
potentially undesired phenotypes. Enriched glial progenitor cells
were thus assessed for their myelinogenic capacity, and were found
able to myelinate shiverer axons (Warrington et al., "Differential
Myelinogenic Capacity of Specific Development Stages of the
Oligodendrocyte Lineage Upon Transplantation Into Hypomyelinating
Hosts," J. Neurosci Res 34:1-13 (1993)), though with low
efficiency, likely due to predominantly astrocytic differentiation
by the grafted cells. Snyder and colleagues (Yandava et al.,
"Global Cell Replacement is Feasible via Neural Stem Cell
Transplantation: Evidence from the Dysmyelinated Shiverer Mouse
Brain," Proc. Natl. Acad. Sci. 96:7029-7034 (1999)) subsequently
noted that immortalized multipotential progenitors could also
contribute to myelination in shiverers. Duncan and colleagues
similarly noted that oligosphere-derived cells raised from the
neonatal rodent subventricular zone could engraft another
dysmyelinated mutant, the myelin-deficient rat, upon perinatal
intraventricular administration (Learish et al., "Intraventricular
Transplantation of Oligodendrocyte Progenitors into a Fetal Myelin
Mutant Results in Widespread Formation of Myelin," Ann Neurol
46:716-722 (1999)). These studies notwithstanding, the ability of
human oligodendrocyte progenitor cell isolates to myelinate
dysmyelinated brain has not hitherto been examined.
[0005] The present invention is directed to overcoming the
deficiencies in the art.
SUMMARY OF THE INVENTION
[0006] One aspect of the present invention is directed to a method
of remyelinating demyelinated axons by treating the demyelinated
axons with oligodendrocyte progenitor cells under conditions which
permit remyelination of the axons.
[0007] Another aspect of the present invention relates to a method
of treating a subject having a condition mediated by a loss of
myelin or a loss of oligodendrocytes by administering to the
subject oligodendrocyte progenitor cells under conditions effective
to treat the condition mediated by a loss of myelin or a loss of
oligodendrocytes.
[0008] A further aspect of the present invention relates to an in
vitro method of identifying and separating oligodendrocyte
progenitor cells from a mixed population containing other mammalian
brain or spinal cord cell types. This method involves removing
neurons and neuronal progenitor cells from the mixed population to
produce a treated mixed population. The oligodendrocyte progenitor
cells are then separated from the treated mixed population to form
an enriched population of oligodendrocyte progenitor cells.
[0009] Applicants have developed means by which glial and
oligodendrocytic progenitor cells may be isolated from the human
brain; this has allowed the use of highly enriched isolates of
native human oligodendrocyte progenitor cells (OPC) for cell
transplantation studies.
[0010] In this study, it was investigated whether highly enriched
populations of glial progenitor cells directly isolated from the
human brain might be used as a substrate for cell-based therapy of
congenital dysmyelination. Specifically, it was postulated that
human OPCs, derived from the fetal brain during its period of
maximum oligoneogenesis, as well as from the adult brain, would be
sufficiently migratory and myelinogenic to mediate the widespread
myelination of a perinatal host. This showed that oligodendrocyte
progenitor cells could indeed be extracted in bulk and isolated via
surface antigen-based FACS from both the fetal and adult human
forebrain. These cells were capable of widespread and
high-efficiency myelination of the shiverer brain after perinatal
xenograft. They infiltrated widely throughout the presumptive white
matter, ensheathed resident murine axons, and formed antigenically
and ultrastructurally compact myelin. After implantation, the cells
slowed their mitotic expansion with time, and generated neither
undesired phenotypes nor parenchymal aggregates. Both fetal and
adult-derived OPCs were competent to remyelinate murine axons, but
important differences were noted: whereas fetal OPCs were highly
migratory, they myelinated slowly and inefficiently. In contrast,
adult-derived OPCs migrated over lesser distances, but they
myelinated more rapidly and in higher proportions than their fetal
counterparts. Thus, these isolates of human glial progenitor cells
may provide effective cellular substrates for remyelinating the
congenitally dys- or hypomyelinated brain. In practical terms, the
choice of stage-defined cell type may be dictated by both the
availability of donor material, and by the specific biology of the
disease target, since both fetal and adult OPCs proved competent to
effect structural remyelination.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIGS. 1A-D show fluorescence-activated sorting of fetal
human oligodendrocyte progenitor cells. This shows the result of
dual-color FACS of a 23 week human fetal ventricular zone
dissociate, after concurrent immunostaining for both A2B5 and
PSA-NCAM. The FACS plot on the left (FIG. 1A) illustrates a matched
but unstained 23 week dissociate. On the right, FIG. 1B shows the
same VZ dissociate, sorted after dual immunolabeling for A2B5 (FL1,
y axis) and PSA-NCAM (FL2, x axis). FIGS. 1C-D show A2B5-sorted
cells expressed the oligodendrocytic sulfatide antigen recognized
by monoclonal antibody O4. The A2B5.sup.+/PSA-NCAM.sup.- fraction
in R1R3, comprising 16.5% of the dissociate, corresponded to glial
progenitor cells. Although these were able to generate both
astrocytes and oligodendrocytes, they were preferentially
oligoneogenic when derived at this gestational age, and were thus
designated as oligodendrocyte progenitor cells (OPCs). In contrast,
the R1R5 fraction, defined by the antigenic phenotype
A2B5.sup.-/PSA-NCAM.sup.+, generated largely neurons in vitro, and
was therefore defined as a neuronal progenitor pool.
[0012] FIGS. 2A-E show fetal human OPCs migrate rapidly to
infiltrate the forebrain. This composite shows the distribution of
transplanted human cells 4 weeks after perinatal implantation into
shiverer recipients. The human cells were localized by anti-human
nuclear antigen (ANA) immunostaining; low-power fluorescence images
were then collected at representative anteroposterior levels and
schematized. The engrafted cells have dispersed widely throughout
the forebrain, although most remain in the subcortical white matter
tracts. FIG. 2A shows a sagittal schematic identifying the levels
sampled. FIGS. 2B-E show sections corresponding to AP 1.25, 1.0,
-1.0, and -2.0, in the coronal plane. Scale bar=3 mm.
[0013] FIGS. 3A-I show engrafted human OPCs myelinate an extensive
region of the forebrain. FIGS. 3A-B show that extensive myelin
basic protein expression by sorted human fetal OPCs, implanted into
homozygote shiverer mice as neonates, indicates that large regions
of the corpus callosum (FIG. 3A and FIG. 3B, 2 different mice) have
myelinated by 12 weeks (MBP). FIG. 3C shows that human OPCs also
migrated to and myelinated fibers throughout the dorsoventral
extents of the internal capsules, manifesting widespread
remyelination of the forebrain after a single perinatal injection.
FIG. 3D demonstrates that myelin basic protein expression, in an
engrafted shiverer callosum 3 months after perinatal xenograft, is
associated with human donor cells, identified by human nuclear
antigen (hNA). Both the engrafted human cells and their associated
myelin were invariably found to lay parallel to callosal axonal
tracts. FIGS. 3E-H show confocal optical sections of implanted
shiverer corpus callosum, with human cells (hNA) surrounded by
myelin basic protein (MBP). Human cells (arrows) are found within
meshwork of MBP.sup.+ fibers (FIG. 3E, merged image of optical
sections FIGS. 3F-H, taken 1 .mu.m apart). FIG. 31 demonstrates
that OPCs were recruited as oligodendrocytes or astrocytes in a
context-dependent manner, such that implanted OPCs typically
matured as myelinogenic oligodendrocytes in the presumptive white
matter, but as GFAP-defined astrocytes in both white and gray and
white matter. This photo shows the striatocallosal border of a
shiverer brain, 3 months after perinatal engraftment with human
fetal OPCs (hNA). Donor-derived MBP expression is evident in the
corpus callosum, while donor-derived GFAP.sup.+ astrocytes
predominate on the striatal side. Scale bar=200 .mu.m. Scale: FIGS.
3A-C, 1 mm; FIG. 3D, 100 .mu.m; FIGS. 3E-H, 20 .mu.m; FIG. 31, 200
.mu.m.
[0014] FIGS. 4A-G show axonal ensheathment and myelin compaction by
engrafted human progenitor cells. FIG. 4A is a confocal micrograph
showing a triple-immunostain for MBP, human ANA, and neurofilament
protein. In this image, all MBP immunostaining is derived from the
sorted human OPCs, whereas the NF.sup.+ axons are those of the
mouse host. Arrows identify segments of murine axons ensheathed by
human oligodendrocytic MBP. FIG. 4B is a 2 .mu.m deep composite of
optical sections, taken through the corpus callosum of a shiverer
recipient sacrificed 12 weeks after fetal OPC implantation.
Shiverer axons were scored as ensheathed when the index lines
intersected an NF.sup.+ axon abutted on each side by
MBP-immunoreactivity. The asterisk indicates the field enlarged in
FIG. 4C. In FIG. 4C, at higher magnification, MBP-immunoreactivity
can be seen to surround ensheathed axons on both sides. FIG. 4D is
an electron micrographs of a sagittal section through the corpus
callosum of an adult shi/shi homozygote. Shiverer axons typically
have a single loose wrapping of myelin that fails to compact, so
that major dense lines fail to form. FIGS. 4E-G are representative
micrographs of 16-week old shiverer homozygotes, implanted with
human oligodendrocyte progenitor cells shortly after birth. These
images show resident shiverer axons with densely compacted myelin
sheaths. The asterisk indicates the field enlarged in the inset.
Inset, Major dense lines are noted between myelin lamellae,
providing EM confirmation of myelination by engrafted human OPCs.
Scale bar=FIG. 4A, 20 .mu.m; FIG. 4B, 40 .mu.m; FIGS. 4C-F, 1
.mu.m.
[0015] FIGS. 5A-C show mitotic activity of engrafted progenitors
falls with time. FIGS. 5A-B show BrdU incorporation by transplanted
fetal human OPCs, at 4 (FIG. 5A) and 12 weeks (FIG. 5B) after
xenograft. The shiverer recipients were given intraventricular
injections of sorted human OPCs on postnatal day 1, then injected
with BrdU (100 .mu.g/g, i.p.) twice daily for 2 days prior to
sacrifice. Mitotic human OPCs were observed as BrdU/hNA.sup.+ cells
(arrows). Scale bar=50 .mu.m. FIG. 5C is a regression of the
incidence of mitotically-active donor cells as a function of time
after perinatal implant. The fraction of human donor cells that
incorporated BrdU during the 48 hrs preceding sacrifice dropped
from 42.+-.6.1% at 4 weeks, to 8.2.+-.2.4% at 12 weeks. Regression
analysis revealed that the rate of BrdU incorporation declined with
time according to the exponential regression: y=83.4e.sup.-0.22x,
with a correlation coefficient of r=-0.87 (p=0.012).
[0016] FIGS. 6A-F show fetal and adult OPCs differed substantially
in their speed and efficiency of myelinogenesis. FIG. 6A shows that
adult-derived human OPCs (hNA) achieved dense MBP expression by 4
weeks after xenograft. In contrast, FIG. 6B shows fetal OPCs
expressed no detectable MBP-IR at 4 weeks, with such expression not
noted until 12 wks. Scale=100 .mu.m. FIGS. 4C-D are low and high
magnification coronal images of the callosal-fimbrial junction of a
shiverer homozygote, showing dense myelination by 12 weeks after
perinatal engraftment with adult-derived hOPCs. When assessed
individually, almost half of the donor cells in this recipient
white matter were found to express MBP. FIG. 6E shows that a
substantially higher proportion of implanted adult OPCs developed
MBP expression then did fetal OPCs, when both were assessed at 12
weeks. FIG. 6F shows that fetal donor cells nonetheless engrafted
more efficiently and in higher numbers than did
identically-implanted adult OPCs. * indicates p<0.05; **
p<0.005, each of Student's t-test (2-tailed). Scale: FIGS. 6A-B,
100 .mu.m, FIG. 6C, 1 mm; FIG. 6D, 30 .mu.m.
DETAILED DESCRIPTION OF THE INVENTION
[0017] As used herein, the term "isolated" when used in conjunction
with a nucleic acid molecule refers to: 1) a nucleic acid molecule
which has been separated from an organism in a substantially
purified form (i.e. substantially free of other substances
originating from that organism), or 2) a nucleic acid molecule
having the same nucleotide sequence but not necessarily separated
from the organism (i.e. synthesized or recombinantly produced
nucleic acid molecules).
[0018] One aspect of the present invention is directed to a method
of remyelinating demyelinated axons by treating the demyelinated
axons with oligodendrocyte progenitor cells under conditions which
permit remyelination of the axons.
[0019] The remyelination of demyelinated axons can be carried out
by:
[0020] (1) transuterine fetal intraventricular injection; (2)
intraventricular or intraparenchymal (i.e. brain, brain stem, or
spinal cord) injections; (3) intraparenchymal injections into adult
and juvenile subjects; or (4) intravascular administration. Such
administration involves cell doses ranging from 1.times.10.sup.5 to
5.times.10.sup.7, depending on the extent of desired
remyelination.
[0021] Another aspect of the present invention relates to a method
of treating a subject having a condition mediated by a loss of
myelin or a loss of oligodendrocytes by administering to the
subject oligodendrocyte progenitor cells under conditions effective
to treat the condition mediated by a loss of myelin or a loss of
oligodendrocytes.
[0022] Conditions mediated by a loss of myelin include an ischemic
demyelination condition, an inflammatory demyelination condition, a
pediatric leukodystrophy, mucopolysaccharidosis, perinatal germinal
matrix hemorrhage, cerebral palsy, periventricular leukoinalacia,
radiation-induced conditions, and subcortical leukoencephalopathy
due to various etiologies.
[0023] Ischemic demyelination conditions include cortical stroke,
Lacunar infarct, post-hypoxic leukoencephalopathy, diabetic
leukoencephalopathy, and hypertensive leukoencephalopathy.
[0024] Inflammatory demyelination conditions include multiple
sclerosis, Schilder's Disease, transverse myelitis, optic neuritis,
post-vaccination encephalomyelitis, and post-infectious
encephalomyelitis.
[0025] Pediatric leukodystrophy conditions include lysosomal
storage diseases (e.g., Tay-Sachs Disease), Cavavan's Disease,
Pelizaens-Merzbacher Disease, and Crabbe's Globoid body
leukodystrophy.
[0026] An example of mucopolysaccharidosis is Sly's Disease.
[0027] Radiation-induced conditions include radiation-induced
leukoencephalopathy and radiation-induced myelitis.
[0028] Etiologies causing subcortical leukoencephalopathy include
HIV/AIDS, head trauma, and multi-infarct states.
[0029] Oligodendrocyte progenitor cells are administered in
accordance with this aspect of the present invention in
substantially the same manner as described above with regard to
treatment of demyelinated axons with oligodendrocyte progenitor
cells.
[0030] In one embodiment of the present invention, oligodendrocyte
progenitor cells are administered to the subject after
administering radiation and before demyelination has occurred. The
purpose of radiation administration is to treat primary and
metastatic tumors of the central nervous system.
[0031] The subject treated with oligodendrocyte progenitor cells in
accordance with the present invention is preferably a human and,
most preferably, an adult or post-natal human.
[0032] A further aspect of the present invention relates to an in
vitro method of identifying and separating oligodendrocyte
progenitor cells from a mixed population containing other mammalian
brain or spinal cord cell types. This method involves removing
neurons and neuronal progenitor cells from the mixed population to
produce a treated mixed population. The oligodendrocyte progenitor
cells are then separated from the treated mixed population to form
an enriched population of oligodendrocyte progenitor cells.
[0033] The step of removing neurons and neuronal progenitor cells
from a mixed population containing other mammalian brain or spinal
cord cell types can be carried out by promoter based cell sorting.
This procedure includes providing a mixed population of cell types
from the brain and spinal cord which population includes neurons
and neuronal progenitor cells as well as oligodendrocyte progenitor
cells and selecting a promoter which functions in the neurons and
neuronal progenitor cells, but not in the oligodendrocyte
progenitor cells. A nucleic acid molecule encoding a marker protein
under control of the promoter is introduced into the mixed
population of cell types, and the population of neurons or neuronal
progenitor cells is allowed to express the marker protein. The
cells expressing the marker protein are separated from the mixed
population of cells, with the separated cells being the neurons and
neuronal progenitor cells. The process of selecting neurons and
neuronal progenitor cells from a mixed population of cell types
using a promoter that functions in the neurons and neuronal
progenitor cells and a nucleic acid encoding a marker protein is
described in U.S. Pat. No. 6,245,564 to Goldman et. al., which is
hereby incorporated by reference in its entirety.
[0034] The neurons and neuronal progenitor cells can be separated
from a mixed population containing other mammalian brain or spinal
cord cell types in accordance with the present invention, as long
as a promoter specific for the chosen cell is available.
"Specific", as used herein to describe a promoter, means that the
promoter functions only in the chosen cell type. A chosen cell type
can refer to different types of cells or different stages in the
developmental cycle of a progenitor cell. For example, the chosen
cell may be committed to a particular adult cell phenotype and the
chosen promoter only functions in that progenitor cell; i.e. the
promoter does not function in adult cells. Although committed and
uncommitted progenitor cells may both be considered progenitor
cells, these cells are at different stages of progenitor cell
development and can be separated according to the present invention
if the chosen promoter is specific to the particular stage of the
progenitor cell. Those of ordinary skill in the art can readily
determine a cell of interest to select based on the availability of
a promoter specific for that cell of interest.
[0035] Suitable promoters which are specific for neurons or
neuronal progenitor cells include a MAP-1B promoter (Liu and
Fischer, Gene 171:307-308 (1996), which is hereby incorporated by
reference in its entirety), an NCAM promoter (Hoist et al., J Biol
Chem 269:22245-22252 (1994), which is hereby incorporated by
reference in its entirety), an HES-5 HLH protein promoter
(Takebayashi et al., J Biol Chem 270:1342-1349 (1995), which is
hereby incorporated by reference in its entirety), an al-tubulin
promoter (Gloster, A., et al., J Neurosci 14:7319-7330 (1994),
which is hereby incorporated by reference in its entirety), an
.alpha.-internexin promoter (Ching et al., J Biol Chem
266:19459-19468 (1991), which is hereby incorporated by reference
in its entirety), and a GAP-43 promoter (Starr et al., Brain Res
638:211-220 (1994), which is hereby incorporated by reference in
its entirety).
[0036] Having determined the promoter specific for the neurons and
neuronal progenitor cells, a nucleic acid molecule encoding a
protein marker, preferably a green fluorescent protein, under the
control of the promoter is introduced into a plurality of cells to
be sorted.
[0037] The isolated nucleic acid molecule encoding a green
fluorescent protein can be deoxyribonucleic acid (DNA) or
ribonucleic acid (RNA, including messenger RNA or mRNA), genomic or
recombinant, biologically isolated or synthetic. The DNA molecule
can be a cDNA molecule, which is a DNA copy of a messenger RNA
(mRNA) encoding the GFP. In one embodiment, the GFP can be from
Aequorea victoria (U.S. Pat. No. 5,491,084 to Prasher et. al.,
which is hereby incorporated by reference in its entirety). A
plasmid designated pGFP10.1 has been deposited pursuant to, and in
satisfaction of, the requirements of the Budapest Treaty on the
International Recognition of the Deposit of Microorganisms for the
Purposes of Patent Procedure, with the American Type Culture
Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852 under
ATCC Accession No. 75547 on Sep. 1, 1993. This plasmid is
commercially available from the ATCC due to the issuance of U.S.
Pat. No. 5,491,084 on Feb. 13, 1996 in which the plasmid is
described. This plasmid comprises a cDNA which encodes a green
fluorescent protein (GFP) of Aequorea victoria as disclosed in U.S.
Pat. No. 5,491,084 to Chalfie et al., which is hereby incorporated
by reference in its entirety. A mutated form of this GFP (a
red-shifted mutant form) designated pRSGFP-C1 is commercially
available from Clontech Laboratories, Inc. (Palo Alto, Calif.).
[0038] The plasmid designated pT.alpha.1-RSGFP has been deposited
pursuant to, and in satisfaction of, the requirements of the
Budapest Treaty on the International Recognition of the Deposit of
Microorganisms for the Purposes of Patent Procedure, with the
American Type Culture Collection (ATCC), 12301 Parklawn Drive,
Rockville, Md. 20852 under ATCC Accession No. 98298 on Jan. 21,
1997.
[0039] This plasmid uses the red shifted GFP (RS-GFP) of Clontech
Laboratories, Inc. (Palo Alto, Calif.), and the T.alpha.1 promoter
sequence provided by Dr. F. Miller (Montreal Neurological
Institute, McGill University, Montreal, Canada). In accordance with
the subject invention, the T.alpha.1 promoter can be replaced with
another specific promoter, and the RS-GFP gene can be replaced with
another form of GFP, by using standard restriction enzymes and
ligation procedures.
[0040] Mutated forms of GFP that emit more strongly than the native
protein, as well as forms of GFP amenable to stable translation in
higher vertebrates, are now available and can be used for the same
purpose. The plasmid designated pT.alpha.1-GFPh has been deposited
pursuant to, and in satisfaction of, the requirements of the
Budapest Treaty on the International Recognition of the Deposit of
Microorganisms for the Purposes of Patent Procedure, with the
American Type Culture Collection (ATCC), 12301 Parklawn Drive,
Rockville, Md. 20852 under ATCC Accession No. 98299 on Jan. 21,
1997. This plasmid uses the humanized GFP (GFPh) of Zolotukhin and
Muzyczka (Levy, J., et al., Nature Biotechnol 14:610-614 (1996),
which is hereby incorporated by reference in its entirety), and the
T.alpha.1 promoter sequence provided by Dr. F. Miller (Montreal).
In accordance with the subject invention, the T.alpha.1 promoter
can be replaced with another specific promoter, and the GFPh gene
can be replaced with another form of GFP, by using standard
restriction enzymes and ligation procedures. Any nucleic acid
molecule encoding a fluorescent form of GFP can be used in
accordance with the subject invention.
[0041] Standard techniques are then used to place the nucleic acid
molecule encoding GFP under the control of the chosen cell specific
promoter. Generally, this involves the use of restriction enzymes
and ligation.
[0042] The resulting construct, which comprises the nucleic acid
molecule encoding the GFP under the control of the selected
promoter (itself a nucleic acid molecule) (with other suitable
regulatory elements if desired), is then introduced into a
plurality of cells which are to be sorted. Techniques for
introducing the nucleic acid molecules of the construct into the
plurality of cells may involve the use of expression vectors which
comprise the nucleic acid molecules. These expression vectors (such
as plasmids and viruses) can then be used to introduce the nucleic
acid molecules into the plurality of cells.
[0043] Various methods are known in the art for introducing nucleic
acid molecules into host cells. These include: 1) microinjection,
in which DNA is injected directly into the nucleus of cells through
fine glass needles; 2) dextran incubation, in which DNA is
incubated with an inert carbohydrate polymer (dextran) to which a
positively charged chemical group (DEAE, for diethylaminoethyl) has
been coupled (the DNA sticks to the DEAE-dextran via its negatively
charged phosphate groups, large DNA-containing particles stick in
turn to the surfaces of cells (which are thought to take them in by
a process known as endocytosis), and some of the DNA evades
destruction in the cytoplasm of the cell and escapes to the
nucleus, where it can be transcribed into RNA like any other gene
in the cell); 3) calcium phosphate coprecipitation, in which cells
efficiently take in DNA in the form of a precipitate with calcium
phosphate; 4) electroporation, in which cells are placed in a
solution containing DNA and subjected to a brief electrical pulse
that causes holes to open transiently in their membranes so that
DNA enters through the holes directly into the cytoplasm, bypassing
the endocytotic vesicles through which they pass in the
DEAE-dextran and calcium phosphate procedures (passage through
these vesicles may sometimes destroy or damage DNA); 5) liposomal
mediated transformation, in which DNA is incorporated into
artificial lipid vesicles, liposomes, which fuse with the cell
membrane, delivering their contents directly into the cytoplasm; 6)
biolistic transformation, in which DNA is absorbed to the surface
of gold particles and fired into cells under high pressure using a
ballistic device; 7) naked DNA insertion; and 8) viral-mediated
transformation, in which nucleic acid molecules are introduced into
cells using viral vectors. Since viral growth depends on the
ability to get the viral genome into cells, viruses have devised
efficient methods for doing so. These viruses include retroviruses,
lentivirus, adenovirus, herpesvirus, and adeno-associated
virus.
[0044] As indicated, some of these methods of transforming a cell
require the use of an intermediate plasmid vector. U.S. Pat. No.
4,237,224 to Cohen and Boyer, which is hereby incorporated by
reference in its entirety, describes the production of expression
systems in the form of recombinant plasmids using restriction
enzyme cleavage and ligation with DNA ligase. These recombinant
plasmids are then introduced by means of transformation and
replicated in unicellular cultures including procaryotic organisms
and eucaryotic cells grown in tissue culture. The DNA sequences are
cloned into the plasmid vector using standard cloning procedures
known in the art, as described by Sambrook et al., Molecular
Cloning: A Laboratory Manual, 2d Edition, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (1989), which is hereby
incorporated by reference in its entirety.
[0045] In accordance with one of the above-described methods, the
nucleic acid molecule encoding the GFP is thus introduced into a
plurality of cells. The promoter which controls expression of the
GFP, however, only functions in the cell of interest. Therefore,
the GFP is only expressed in the cell of interest. Since GFP is a
fluorescent protein, the cells of interest can therefore be
identified from among the plurality of cells by the fluorescence of
the GFP.
[0046] Any suitable means of detecting the fluorescent cells can be
used. The cells may be identified using epifluorescence optics, and
can be physically picked up and brought together by Laser Tweezers
(Cell Robotics Inc., Albuquerque, N. Mex.). They can be separated
in bulk through fluorescence activated cell sorting, a method that
effectively separates the fluorescent cells from the
non-fluorescent cells.
[0047] One embodiment of separating oligodendrocyte progenitor
cells from the treated mixed population, in accordance with this
aspect of the present invention, is carried out by promoter based
cell separation as described above, except that rather than
starting with the introduction of a nucleic acid molecule encoding
a fluorescent protein under control of the promoter into the entire
mixed population containing mammalian brain or spinal cord cell
types besides the oligodendrocyte progenitor cells, that nucleic
acid molecule is introduced into the treated mixed population. In
sorting out oligodendrocyte progenitor cells from the treated mixed
population, a promoter specific for oligodendrocyte progenitor
cells is utilized. The promoter suitable for carrying out this
aspect of the present invention can be a cyclic nucleotide
phosphorylase I promoter, a myelin basic protein promoter, a JC
virus minimal core promoter, a proteolipid protein promoter, a qk1
promoter (i.e. the promoter for the quaking gene product), and a
cyclic nucleotide phosphorylase II promoter.
[0048] As an alternative to using promoter-based cell sorting to
recover oligodendrocyte progenitor cells from the treated mixed
population, an immunoseparation procedure is utilized.
[0049] This involves separating cells based on proteinaceous
surface markers naturally present on progenitor cells of a specific
type. For example, the surface marker A2B5 is an initially
expressed early oligodendrocyte marker. See Nunes et al.,
"Identification and Isolation of Multipotential Neural Progenitor
Cells from the Adult Human White Matter, " Soc. Neurosci. Abstr.
(2001), which is hereby incorporated by reference. Using an
antibody specific to that marker oligodendrocyte progenitor cells
can be separated from a mixed population of cell types. Such
antibodies can be labeled with a fluorescent tag to facilitate
separation of cells to which they bind. Alternatively, the
antibodies can be attached to paramagnetic beads so that cells
which bind to the beads through the attached antibodies can be
recovered by a biomagnetic separation process.
[0050] A hybridoma producing monoclonal antibodies specific to Gq
ganglioside, designated A2B5 has been deposited pursuant to, and in
satisfaction of, the requirements of the Budapest Treaty on the
International Recognition of the Deposit of Microorganisms for the
Purposes of Patent Procedure, with the American Type Culture
Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852 under
ATCC Accession No. CRL-01520.
[0051] The enriched population of oligodendroctye progenitor cells
is at least 90% pure, preferably at least 95% pure, and most
preferably at least 99% pure. The mixed population of cell types
used to carry out this aspect of the present invention are
preferably human cells. These cells are desirably adult or
post-natal human cells.
[0052] Instead of utilizing the above-described procedure of
obtaining oligodendrocyte progenitor cells by removing nerons and
neuronal progenitor cells from a mixed population of brain and
spinal cord cell types, leaving a treated mixed population and then
separating the oligodendrocyte progenitor cells from the treated
population, the oligodendrocyte progenitor cells can be recovered
directly from the mixed population of brain and spinal cord cell
types using promoter based cell sorting as described in U.S. Pat.
No. 6,245,564 to Goldman, et. al., and U.S. patent application Ser.
No. 09/282,239 to Goldman et. al., which are hereby incorporated by
reference in their entirety. This method is essentially as
described above, using a promoter which functions only in
oligodendrocyte progenitor cells.
EXAMPLES
Example 1
Cells
[0053] Cells Tissue from late gestational age human fetuses (21 to
23 weeks) were obtained at abortion. The forebrain
ventricular/subventricular zones were rapidly dissected free of the
remaining brain parenchyma, and the samples chilled on ice. The
minced samples were then dissociated using papain/DNAse as
previously described
[0054] (Keyoung et al., "Specific Identification, Selection and
Extraction of Neural Stem Cells from the Fetal Human Brain," Nature
Biotechnology 19:843-850 (2001), which is hereby incorporated by
reference in its entirety), always within 3 hours of extraction.
The dissociates were then maintained overnight in minimal culture
media of DMEM/F12/N1 with 20 ng/ml FGF.
Example 2
Sorting
[0055] The day after dissociation, the cells were incubated 1:1
with MAb A2B5 supernatant (clone 105; ATCC, Manassas, Va.), for 30
minutes, then washed and labeled with microbead-tagged rat
anti-mouse IgM (Miltenyi Biotech). All incubations were done at
4.degree. C. on a rocker. In some instances, 2-channel
fluorescence-activated cell sorting was done to define the
proportions and phenotypic homogeneity of A2B5 and PSA-NCAM-defined
subpopulations, using a FACSVantage SE/Turbo, according to
previously described methods (Keyoung et al., "Specific
Identification, Selection and Extraction of Neural Stem Cells from
the Fetal Human Brain," Nature Biotechnology 19:843-850 (2001) and
Roy et al., "In Vitro Neurogenesis by Progenitor Cells Isolated
from the Adult Human Hippocampus," Nat Med 6:271-277 (2000), which
are hereby incorporated by reference in their entirety). More
typically, and for all preparative sorts for transplant purposes,
magnetic separation of A2B5.sup.+ cells (MACS; Miltenyi) was next
performed, following the manufacturer's protocol. The bound cells
were then eluted and incubated with anti-NCAM (Pharmingen) at 1:25
for 30 minutes, and labeled with anti-mouse PE at 1:200. The
PSA-NCAM.sup.+ population was then removed by FACS, leaving a
highly enriched population of A2B5.sup.+/PSA-NCAM.sup.- cells.
These were maintained in vitro for 1-7 days in base media with 20
ng/ml bFGF, until implantation. See FIG. 1.
Example 3
Transplantation and Tagging
[0056] Homozygous shiverers were bred in a colony. Within a day of
birth, the pups were cryoanesthetized for cell delivery. The donor
cells were then implanted using a pulled glass pipette inserted
through the skull, into either the corpus callosum, the internal
capsule, or the lateral ventricle. The pups were then returned to
their mother, and later killed after 4, 8, 12, or 16 weeks. For
some experiments, recipient mice were injected for 2 days before
sacrifice with BrdU (100 .mu.g/g, as a 1.5 mg/100 .mu.l solution),
q12 hrs for 2 consecutive days.
Example 4
Immunohistochemistry
[0057] The transplanted cells were identified using anti-human
nuclei antibody from Chemicon (MAB 1281), and either Rhodamine Red
X-conjugated goat anti-mouse (Jackson; cat. 115-295-146) or
unconjugated rabbit anti-mouse Fab (Jackson 315-007-003) followed
by Rhodamine Red X-goat anti-rabbit (Jackson 111-295-144). See FIG.
2. CNP was recognized using Sternberger Monoclonal 91, MBP by
either Sternberger MAb 94 or Abcam 7349 (rat); human GFAP was
detected using anti-human GFAP (Sternberger MAb 21). See FIG. 3 and
FIGS. 4A-C. BrdU was immunolabeled concurrently with phenotypic
markers as described (Louissaint et al., "Coordinated Interaction
of Angiogenesis and Neurogenesis in the Adult Songbird Brain,"
Neuron 34:945-960 (2002), which is hereby incorporated by reference
in its entirety).
Example 5
Electron Microscopy
[0058] Animals were perfused with 4% paraformaldehyde and 0.25%
glutaraldehyde in 6% sucrose phosphate buffer (sucrose-PB),
post-fixed in the same solution, then sliced by Vibratome in
alternating thick (400 .mu.m) and thin (100 .mu.m) sections. The
thin sections were immunostained for MBP, while the thick sections
were post-fixed in 2% paraformaldehyde and 2.5% glutaraldehye in
sucrose-PB. Those thick sections adjacent to thin sections
exhibiting overt MBP expression were then processed in 1%
osmium-1.5% ferricyanide, 1.5% aqueous uranyl acetate, dehydrated
through propylene oxide, then embedded in Epon and stained with
lead citrate. See FIGS. 4D-G.
Example 6
Oligodendrocyte Progenitors Can Be Sorted From the Fetal Human
Ventricular Wall
[0059] Cells dissociated from the late second trimester human
ventricular zone of 21-23 weeks gestation were first magnetically
sorted to isolate A2B5.sup.+ cells. These included both
oligodendrocyte and neuronal progenitor cells. Since PSA-NCAM is
expressed by virtually all immature neurons at this stage of human
ventricular zone development, FACS was then used to select out
PSA-NCAM.sup.+ cells from the larger A2B5.sup.+ cell population.
This removal from the A2B5.sup.+ pool of NCAM-defined neuronal
progenitor cells and young neurons yielded a subpopulation of
A2B5.sup.+/PSA-NCAM.sup.- cells, that defined our oligodendrocyte
progenitor pool. By two-color fluorescence-activated cell sorting
(FACS), with high-stringency control thresholds intended to limit
the incidence of false positives to <0.1%, it was determined
that the A2B5.sup.+/PSA-NCAM.sup.- fraction constituted
15.4.+-.4.8% of the cells in these pooled 21-23 week ventricular
vvzone samples (n=5) (FIG. 1). The glial restriction and
oligodendrocytic bias of these VZ progenitors was verified in
vitro, as the PSA-NCAM-depleted A2B5.sup.+ pool generated largely
oligodendrocytes--and exclusively glia--under basal culture
conditions (FIG. 1). Under these same conditions, the
PSA-NCAM.sup.+ fraction of A2B5.sup.+ cells differentiated
predominantly into neurons. Thus, two distinct methods of dual
antigen immunosorting, two-color FACS and serial immunomagnetic
enrichment of A2B5.sup.+ cells followed by FACS depletion of
PSA-NCAM.sup.+ cells, each permitted the selective enrichment and
high-yield extraction of oligodendrocyte progenitor cells from the
21-23 week fetal human ventricular zone. Since the latter
technique--immunomagnetic separation followed by single-color FACS
depletion--achieved higher net yields than direct two-color FACS,
this serial approach was used for extracting and isolating the
engrafted human OPC populations.
Example 7
Implanted Oligodendrocyte Progenitors Migrated Widely After
Xenograft
[0060] Homozygote shi/shi mice were injected intraventricularly and
intracallosally with progenitor cell isolates at P0-1. The animals
were divided into subgroups that were sacrificed thereafter at 4
week intervals, at 4, 8, 12, or 16 weeks of age. None of the
animals were immunosuppressed; perinatal tolerization was relied on
to ensure graft acceptance, as a result of which animals were
transplanted on either their day of birth or the day after (P0-1),
but not beyond. These injections yielded significant and
quantifiable cell engraftment (defined as.gtoreq.100 cells per
coronal section at 3 rostrocaudal levels, sampled>100 .mu.m
apart), in 34 of the 44 neonatal mice injected for this study (25
of 33 injected with fetal hOPCs, and 9 of 11 injected with
adult-derived OPCs). Although aggregates of cells were often noted
in the ventricle at 4 weeks, by 12 weeks most if not all implanted
cells had penetrated the callosal and fimbrial walls to invade the
callosum, fimbria and capsular white matter (FIG. 2).
[0061] The OPCs typically migrated rapidly, dispersing throughout
the subcortical parenchyma from the frontal white matter of the
forceps minor rostrally, to the basis pontis caudally. At 4 weeks,
the implanted cells, identified by their expression of human
nuclear antigen (hNA), were found dispersed widely throughout the
white matter, primarily within the corpus callosum, external
capsule, and fimbria of the hippocampus (FIG. 2). Many nuclei,
especially rostral or caudal to the injection site, appeared
elongated in the orientation of the tracts, with the morphology of
migrants. In addition, a distinct minority entered gray matter
regions, including the septum, striatum, and olfactory bulb, and
less so the neocortex. By 8 weeks, human cells extended widely
throughout the forebrain, and in lesser numbers to the
diencephalon. In 2 of these 8-week animals, cells were noted to
enter the brainstem white matter tracts, traveling through the
cerebral preduncles as far as the basis pontis. In animals allowed
to survive for 12 weeks, cells were noted throughout the forebrain,
though still primarily within the white matter tracts. Although
human nuclei were found both throughout the forebrain, and
scattered about the rostral brainstem, xenograft density was
invariably greatest in the fimbrial and callosal sites of cell
introduction.
Example 8
Engrafted Fetal Progenitors Matured to Express Myelin Basic
Protein
[0062] The next question was whether engrafted fetal-derived
progenitors matured as myelinogenic oligodendrocytes in vivo. To
this end, both implanted and unimplanted control mice were
immunostained for oligodendrocytic myelin basic protein (MBP), at
4, 8, and 12 weeks after implantation. Since shiverer mice express
only the first exon of the MBP gene (Roach, et al., "Chromosomal
Mapping of Mouse Myelin Basic Protein Gene and Structure and
Transcription of the Partially Deleted Gene in Shiverer Mutant
Mice," Cell 42:149-155 (1985), which is hereby incorporated by
reference in its entirety). C-terminal-directed anti-MBP antibodies
do not recognize the truncated MBP of shiverer homozygotes. As a
result, any MBP immunoreactivity detected in transplanted animals
necessarily derives from donor-derived oligodendrocytes. At 4
weeks, no detectable MBP was noted in 10 of 11 animals, despite
widespread cell dispersion; sparse regions of nascent
MBP-immunoreactivity were noted in one mouse. At 8 weeks, patchy
foci of MBP expression were noted in 4 of 7 mice, typically within
their callosa and hippocampal commissures. By 12 weeks though,
widespread MBP expression was noted throughout the forebrain white
matter tracts in 5 of 7 mice. MBP expression was particularly
abundant in the fimbria posteriorly and corpus callosum
anteriorly.
[0063] Indeed, the corpus callosum typically expressed MBP
throughout its mediolateral extent, and along its entire length in
the sagittal plane (FIGS. 3A-C).
[0064] The broad distribution of myelinogenesis by engrafted cells
resulted in a significant volume of myelin reconstitution in the
recipient brains. For instance, in the 12-week brain shown in FIG.
3, the region of callosal myelination extended about 4 mm
rostrocaudally, the length of the corpus callosum, while expanding
as a trigone from a mediolateral width of 1 mm caudally to 4 mm
rostrally. Given an average callosal depth of 200 .mu.m, the
effective volume of MBP-defined myelin production was 1.4 mm.sup.3.
Importantly, this MBP was associated with human donor cells (FIG.
3D). To prove that MBP-IR was exclusively associated with the
implanted human donor OPCs, confocal imaging was used to examine
the co-localization of MBP-immunoreactivity and human nuclear
antigen. Using optical sectioning with orthogonal reconstruction,
it was confirmed that the MBP.sup.+ cells were of human origin, in
that each was associated with a human soma, as defined by
anti-human nuclear immunostaining (FIGS. 3E-H).
Example 9
Progenitor-Derived Oligodendrocytes Remyelinate Axons
[0065] The next issue was whether the donor-derived myelin actually
ensheathed host shiverer axons. To this end, both confocal imaging
and electron microscopy were used to assess axonal ensheathment and
myelin compaction, respectively. Confocal analysis was first done
on a sample of 3 shiverer brains that were each implanted on P1
with 100,000 sorted fetal human OPCs, and then sacrificed at 12
weeks (FIGS. 4A-C). Regions of callosal MBP expression were first
identified by immunolabeling fixed sections. These foci of dense
MBP expression were then assessed by confocal imaging after
immunolabeling for both human nuclear antigen and neurofilament
protein, so as to tag donor-derived cells and host shiverer axons,
respectively. By this means, human progenitors were found to have
generated myelinating oligodendrocytes in great numbers. The myelin
sheaths of these cells were found to be in direct apposition to,
and generally completely surrounded, host axons in their immediate
vicinity. Among the recipients scored, 11.9.+-.1.6% (mean.+-.SE) of
NF.sup.+ host callosal axons were found to be surrounded by
MBP-immunoreactivity (n=3 mice, with 3 fields scored/animal) (FIGS.
4A-C). Sampling was biased to regions of maximal callosal MBP
expression, so that these numbers do not necessarily reflect the
incidence of myelination in all forebrain tracts. Rather, these
data simply confirm that a significant fraction of resident murine
axons may be ensheathed by human myelin following perinatal
engraftment of donor progenitor cells.
[0066] Next, electron microscopy was used to verify that host axons
were actually ensheathed by donor-derived oligodendrocytes, and
that the latter generated ultrastructurally-compact myelin. Since
MBP is required for compacting consecutive layers of myelin
together, its expression is required for formation of the major
dense line of healthy central myelin. In the MBP-deficient
shiverer, myelin is only loosely wrapped around axons, fails to
exhibit more than a few wrappings, and lacks a major dense line. It
was found that in the shi/shi homozygote recipients of perinatal
human progenitor cell transplants, the transplanted human OPCs
indeed not only myelinated, but produced compact myelin with major
dense lines (FIGS. 4D-G). When assessed ultrastructurally at both
12 and 16 weeks after implant, the donor-derived myelin was
confirmed to surround and ensheath host shiverer axons (FIGS.
4D-G).
[0067] This ultrastructural analysis allowed quantification of the
proportion of axons myelinated by donor-derived OPCs, as a means of
validating the data acquired by confocal analysis. In a sample of
MBP.sup.+ fields (n=50), derived from 2 mice implanted on postnatal
day 1 and sacrificed for histology 16 weeks later, an overall
average of 7.4% of resident callosal axons were found to have
donor-derived myelin sheaths (136 of 1832 scored axons), as defined
by the presence of major dense lines. As in the confocal analysis,
these data reflect the net efficiency of myelination achieved in
callosal regions selected on the basis of their
MBP-immunoreactivity, and hence defined up-front as areas of
successful engraftment; the results are not intended to reflect an
unbiased sample of the recipient white matter. That caveat
notwithstanding, these findings demonstrate that sorted fetal human
OPCs can efficiently differentiate as myelinogenic oligodendrocytes
upon perinatal xenograft.
Example 10
The Proportion of Mitotically Active Donor OPCs Slowly Declined
After Xenograft
[0068] The next issue was whether implanted OPCs continued to
divide after engraftment, and if so, for how long. To this end,
mice were implanted with fetal hOPCs at birth (n=6), and then
injected them with BrdU twice a day for two days prior to their
terminal sacrifice, at 4, 8, and 12 weeks of age. Immunostaining
for BrdU revealed that an average of 42.+-.6.1% of engrafted human
OPCs, implanted on the first postnatal day and defined by their
expression of anti-human nuclear antigen (hNA), were still actively
dividing at 4 weeks of age (FIG. 5). In contrast, by 8 and 12 weeks
after implantation, the fraction of mitotic BrdU.sup.+/hNA.sup.+
cells among the engrafted OPCs fell to 11.2.+-.1.6 and 8.2.+-.2.4%,
respectively. These results suggested that the implanted progenitor
cells were initially mitotically active for at least the first
month after engraftment, but then slowed their mitotic activity
thereafter, such that less than 10% of all OPCs and progeny thereof
were demonstrably cycling by their third month post-implant (FIGS.
5A-C). Regression analysis revealed a strong inverse correlation
between the mitotic index of donor-derived cells and the length of
time post-engraftment (r=0.90; p<0.05). Importantly, despite the
preserved mitotic competence of the implanted progenitor pool, no
histologic evidence of tumor formation, anaplasia, or malignant
transformation was noted as long as 3 months after implantation in
any of the fetal OPC-implanted mice of this study (n=34; including
9 analyzed at 16 weeks).
Example 11
Many of the Transplanted Cells Differentiated as Astrocytes
[0069] Some transplanted fetal OPCs differentiated as astrocytes,
as defined by GFAP, and were noted to do so as early as 4 weeks
after implantation. These GFAP.sup.+ astrocytes were found
intermingled with MBP.sup.+ oligodendrocytes, although they
typically extended over a wider area than their oligodendrocytic
counterparts, which were typically restricted to white matter.
Importantly, the implanted fetal hOPCs rarely differentiated as
neurons in the shiverer brain: No heterotopic .beta.III-tubulin or
MAP2-defined neurons were noted in implanted shiverer white matter
at either 4, 8, or 12 weeks after implantation (n=33 total).
Similarly, those cells that migrated to the septum or the striatum
did not differentiate as neurons; neither did the occasional
migrants that were found to enter the dorsal neocortex from the
corpus callosum. Only in 2 mice, in which
hNA/.beta.III-tubulin.sup.+ neurons were found in the olfactory
bulb at 4 weeks, were any human donor-derived neurons noted, likely
reflecting the particularly neurogenic environment of the olfactory
subependyma and bulb. More typically, those donor OPCs that invaded
the gray matter typically developed as astrocytes. As a result, the
donor-derived astrocytes and oligodendrocytes were typically found
in sharply-demarcated geographic domains that corresponded to gray
and white matter, respectively. While donor-derived astrocytes were
typically more abundant in host gray matter, they were nonetheless
dispersed in both gray and white matter; in contrast, donor-derived
oligodendrocytes were excluded from the host gray matter (FIG. 31).
This segregation of donor-derived glial phenotypes led to sharply
defined domain boundaries for the engrafted cells.
Example 12
Adult-Derived OPCs Myelinate More Rapidly Than Fetal OPCs
[0070] Applicants next asked if fetal OPCs differed from their
counterparts derived from the adult human brain, with respect to
either their migration competence, myelinogenic capacity, or time
courses thereof. To this end, 2 litters of shiverer mice were
implanted on P0 with A2B5-sorted adult OPCs (n=12 mice, of whom 9
exhibited successful donor engraftment). These adult-derived hOPCs
were extracted from surgical resections of normal human subcortical
white matter, from which A2B5.sup.+ OPCs were extracted via
A2B5-directed immunomagnetic sorting (IMS), and then cultured
overnight in minimal media prior to their perinatal xenograft. The
implanted mice were allowed to survive for either 4, 8, or 12
weeks, then sacrificed for histology. Their brains were sectioned
and stained for MBP, GFAP and anti-human nuclear antigen, as had
been their fetal OPC-implanted counterparts.
[0071] It was found that fetal and adult-derived human
oligodendrocyte progenitor cells differed substantially in their
respective time courses and efficacy of myelinogenesis upon
xenograft. Adult OPCs myelinated shiverer brain more rapidly than
their fetal counterparts, achieving widespread and dense MBP
expression by 4 weeks after xenograft. In contrast, substantial MBP
expression by fetal OPCs was generally not observed until 12 weeks
post-implant (FIGS. 6A-D).
Example 13
Adult OPCs Produce Myelinogenic Oligodendrocytes with Higher
Efficiency Than Fetal OPCs
[0072] Besides maturing more quickly than fetal OPCs, adult OPCs
were found to give rise to oligodendrocytes in much higher relative
proportions, and with much less astrocytic co-generation, than did
fetal-derived OPCs. When assessed at the midline of the recipient
corpus callosum, 10.2.+-.4.4% of fetal hNA-defined OPCs expressed
MBP at 12 weeks, while virtually none did so at 4 weeks. In
contrast, 39.5 .+-.16.3% of adult OPCs expressed MBP by 4 weeks
after xenograft into matched recipients (p<0.001 by Student's
2-tailed t-test) (See FIG. 6E). Yet substantially higher numbers of
fetal donor cells were found in the host brains, compared to
identically-implanted adult OPCs (see FIG. 6F). Thus, fetal OPCs
engrafted into shiverer recipients as well or better than adult
OPCs, but those adult cells that did engraft were at least
four-times more likely to mature as oligodendrocytes and develop
myelin than their fetal counterparts.
[0073] Moreover while adult OPCs largely remained restricted to the
host white matter, within which they generated almost entirely
MBP.sup.+ oligodendrocytes, fetal OPCs migrated into both gray and
white matter, generating both astrocytes and oligodendrocytes in a
context-dependent manner (FIG. 31). Perhaps as a result of their
greater speed and efficiency of oligodendrocytic differentiation,
implanted adult OPCs and their derivatives rarely migrated beyond
the bounds of the white matter, while fetal OPCs migrated widely,
with their astrocytic and undifferentiated derivatives extending
throughout both the forebrain gray and white matter.
[0074] It has thus been shown that highly enriched isolates of
human OPCs, sorted from the highly oligoneogenic late second
trimester forebrain, can successfully engraft and myelinate the
shiverer mouse brain, a genetic model of perinatal leukodystrophy.
Specifically, it was found that human OPCs may be selectively
extracted from the late second trimester human ventricular zone in
high-yield, using FACS directed at the antigenic phenotype
A2B5.sup.+/PSA-NCAM.sup.-. When implanted to the neonatal murine
forebrain, these cells reliably migrated widely throughout the
forebrain, maturing in the developing white matter as both
oligodendrocytes and astrocytes, and in the presumptive gray matter
as astrocytes. Over a period of 4-12 weeks thereafter, the time
course depending upon whether the implanted human OPCs were of
fetal or adult origin, the donor-derived oligodendrocytes matured
to produce myelin, which led to the widespread myelination of
resident axons within the shiverer subcortex. This myelination,
verified as such by both confocal and ultrastructural analysis, was
geographically extensive, and extended throughout all white matter
regions of the telencephalon.
Example 14
High-Yield Purification of Native Human Forebrain OPCs
[0075] Applicants had previously found that FACS based upon GFP
expression driven by the early oligodendrocytic CNP2 promoter could
be used to isolate oligodendrocyte progenitor cells from the adult
human white matter (Roy et al., "Identification, Isolation, and
Promoter-Defined Separation of Mitotic Oligodendrocyte Progenitor
Cells from the Adult Human Subcortical White Matter," Neurosci
19:9986-9995 (1996), which is hereby incorporated by reference in
its entirety). These cells expressed the surface ganglioside
recognized by the A2B5 antibody, which could also be used to
selectively extract the population from the adult white matter
(Windrem et al., "Progenitor Cells Derived from the Adult Human
Subcortical White Matter Disperse and Differentiate as
Oligodendrocytes Within Demyelinated Regions of the Rat Brain," J.
Neurosci. Res. 69:966-975 (2002), which is hereby incorporated by
reference in its entirety). However, A2B5 recognizes young neurons
as well as oligodendrocytes (Eisenbarth et al., "Monoclonal
Antibody to a Plasma Membrane Antigen of Neurons," Proc. Natl.
Acad. Sci. 76:4913-17 (1979) and Raff et al., "Two Types of
Astrocytes in Cultures of the Developing Rat White Matter:
Differences in Morphology, Surface Gangliosides, and Growth
Characteristics," J. Neurosci. 3:1289-1300 (1983), which are hereby
incorporated by reference in their entirety). Thus, although
A2B5-based separation may be effectively used to extract OPCs from
the adult white matter, which is largely free of neurons, it is not
adequate for doing so from fetal brain, in which A2B5.sup.+ neurons
are abundant. To address this issue, applicants double-sorted
against both A2B5 and polysialylated N-CAM (PSA-NCAM), which is
ubiquitously expressed by young neurons. By excluding
PSA-NCAM.sup.+ cells from the A2B5-sorted sample, a population of
cells that gave rise almost exclusively to glia and principally to
oligodendrocytes was isolated. This A2B5.sup.+/PSA-NCAM.sup.-
phenotype reliably identified an abundant pool of mitotic
oligodendrocyte progenitors in the fetal human brain, which
appeared analogous to the adult progenitor pool recognized by
P/CNP2:hGFP and A2B5 alone. The combination of this high-yield
technique for high-grade enrichment of OPCs, combined with the
great abundance of OPCs in the highly oligoneogenic 21-23 weeks
human ventricular zone, provided for the first time significant
quantities of human oligodendrocyte progenitor cells, isolated in a
purity and quality appropriate for therapeutic implantation.
Example 15
Differential Dispersion During Migration
[0076] In these experiments, highly-enriched pools of human OPCs
were implanted into the brains of neonatal shiverer mice to assess
their migratory activity, oligodendrocytic maturation, and
efficiency of myelinogenesis. It was found that the sorted OPCs
proved highly migratory, and reached most structures of the
forebrain within 4-8 weeks of implantation (FIG. 2). Yet the
dispersal patterns of their two derivative phenotypes,
oligodendrocytes and astrocytes, differed considerably in their
shiverer hosts. Whereas oligodendrocytes were abundant closer to
the injection site, astrocytes dispersed more widely, broadly
invading the forebrain gray matter. This may have reflected a
selection process, with astroglia migrating more rapidly or
aggressively than their oligodendrocytic counterparts. Similarly,
the A2B5.sup.+/PSA-NCAM.sup.- defined pool may be heterogeneous,
such that lineage-restricted oligodendrocyte progenitors may remain
near the site of injection, while less differentiated, more motile
progenitors might continue to migrate during early expansion,
differentiating preferentially as astrocytes upon the cessation of
migration. Alternatively, the preferential migration of astroglia
to gray matter parenchymal sites may reflect a geographic
restriction against oligodendrocytic infiltration beyond the white
matter compartment. It is likely that each of these considerations
contributes to the different dispersion patterns noted.
Example 16
Persistence of Uncommitted Progenitors
[0077] At all timepoints sampled, large numbers of
nestin.sup.+/hNA.sup.+ cells were noted that failed to express
either astrocytic or oligodendrocytic antigens, and which instead
seemed to remain in the host parenchyma as persistent progenitors.
The incidence of these uncommitted
nestin.sup.+/GFAP.sup.-/MBP.sup.- donor cells was clearly higher in
the fetal than adult-derived grafts. Nonetheless, while most
adult-derived OPCs matured as oligodendrocytes, or less so
astrocytes, a large fraction remained
nestin.sup.+/GFAP.sup.-/MBP.sup.- (FIG. 4A). Such uncommitted cells
may constitute both a blessing and a curse in an engrafted
recipient--they likely comprise a source of progenitors that can be
further stimulated in vivo, whether pharmacologically or in
response to demyelinative injury, to give rise to myelinogenic
oligodendrocytes. On the other hand, they might also represent a
potential source of ectopic neurons upon redirection to a neuronal
fate; conceivably, they might also constitute a reservoir of
mitotically competent cells for later neoplastic transformation
(that being said, applicants have never noted tumor formation in
any recipient of human brain-derived progenitor cells). Thus, the
persistence in engrafted recipients of uncommitted progenitors,
whose phenotypic fate and potential for later expansion remain
unclear, provides a cautionary note that must be considered prior
to any use of sorted oligodendrocyte progenitor cells in clinical
therapeutics.
Example 17
Clinical Utility
[0078] The above results suggest that congenital dysmyelination,
like adult demyelination (Windrem et al., "Progenitor Cells Derived
from the Adult Human Subcortical White Matter Disperse and
Differentiate as Oligodendrocytes Within Demyelinated Regions of
the Rat Brain," J. Neurosci. Res. 69:966-975 (2002), which is
hereby incorporated by reference in its entirety), may be an
appropriate target for cell-based therapy, using allografts of
directly isolated human CNS progenitor cells. In the present study,
the effect of donor engraftment and myelination upon either the
disease phenotype or survival of the recipient mice was not
assessed. However, since the shiverer CNS is dysmyelinated
throughout its CNS, it is likely that broad myelination of the
brainstem and spinal cord, as well as of the brain, will be
required for significant therapeutic benefit. Such widespread
graft-associated myelination may require higher cell doses than
those used in this study, delivered at multiple injection sites
spanning the neuraxis. In this regard, the concurrent injection of
higher cell doses into both the cisterna magna and forebrain
ventricles may yield substantially wider donor cell engraftment and
myelinogenesis than achievable through forebrain injection alone
(Mitome et al., "Towards the Reconstruction of Central Nervous
System White Matter Using Precursor Cells," Brain 124:2147-2161
(2001), which is hereby incorporated by reference in its
entirety).
[0079] Such a strategy of cell-based myelination of a dysmyelinated
host might be of special benefit when directed at newborn
recipients, given the immunological tolerance to alloantigens
introduced to neonatal recipients (Ridge et al., "Neonatal
Tolerance Revisited: Turning on Newborn T Cells With Dendritic
Cells," Science 271:1723-1726 (1996); Roser, B., "Cellular
Mechanisms in Neonatal and Adult Tolerance," Immunol. Rev.
107:179-202 (1989); and Witzke et al., "Induction of Tolerance to
Alloantigen," Rev. Immunogenet. 1:374-386 (1999), which are hereby
incorporated by reference in their entirety). None of the animals
received immunosuppressive therapy, and there was no evidence of
immune rejection of the engrafted human cells. This was in marked
contrast to implantation of human OPCs to the adult rat brain,
where immune rejection of implanted cells was a sufficient problem
to mandate high-dose sustained immunosuppression using cyclosporin
(Windrem et al., "Progenitor Cells Derived from the Adult Human
Subcortical White Matter Disperse and Differentiate as
Oligodendrocytes Within Demyelinated Regions of the Rat Brain," J.
Neurosci. Res. 69:966-975 (2002), which is hereby incorporated by
reference in its entirety). As such, congenital diseases such as
the hereditary leukodystrophies, including Krabbe's, Canavan's and
Tay-Sach's among others, as well as perinatal germinal matrix
hemorrhages and the cerebral palsies, may all prove viable targets
for cell-based therapeutic remyelination.
Example 18
Distinct Features of Fetal and Adult Progenitors
[0080] It was surprising to discover that fetal and adult
oligodendrocyte progenitor cells differed fundamentally in their
time course and efficiency of myelinogenesis (FIG. 6).
Adult-derived OPCs were able to mature and myelinate much more
quickly, and with higher efficiency and in greater relative
proportions, than their analogously isolated fetal counterparts.
Whereas fetal OPCs were generally not observed to myelinate until 8
weeks after implant, and to not exhibit substantial myelination
before 12 weeks, adult OPCs matured and myelinated quickly--almost
invariably by 4 weeks. Besides myelinating much more rapidly than
their fetal counterparts, adult OPCs matured as myelinogenic
oligodendrocytes with much higher efficiency--that is, in much
higher relative proportions, and with much less astrocytic
co-generation--than fetal-derived progenitors. As a result of their
more efficient, rapid, and robust myelination, adult-derived OPCs
might appear to constitute a more immediately useful therapeutic
vector than the otherwise analogous, and similarly-derived
fetal-derived OPCs. This observation has significant implications
with regards to the therapeutic application of these cells, most
particularly in regards to the disease targets that one might
choose to approach with fetal and adult OPCs. Fetal cells might be
appropriate therapeutic vectors for preventing dysmyelination in
developing brains otherwise destined for congenital dysmyelination,
in which endogenous myelination is both slow and delayed. In
contrast, diseases of acquired demyelination, in which extant
myelin is lost and mature axons denuded, may require the rapid
maturation and myelination offered by adult-derived
progenitors.
[0081] Thus, human oligodendrocyte progenitor cells may be isolated
from both the fetal and adult human brain, each in a purity and
yield that permit engraftment for the purpose of therapeutic
remyelination. Fetal and adult-derived phenotypes differ, in that
whereas fetal OPCs migrate more extensively, adult OPCs generate
myelin more rapidly, and with less adventitious astrocytic
production. Thus, the two stage-defined phenotypes may prove suited
to quite distinct disease targets and therapeutic strategies.
Nonetheless, both fetal and adult-derived purified human OPCs may
be used to achieve widespread and efficient myelination of the
congenitally dysmyelinated mammalian brain.
[0082] Although the invention has been described in detail for the
purpose of illustration, it is understood that such detail is
solely for that purpose, and variations can be made therein by
those skilled in the art without departing from the spirit and
scope of the invention which is defined by the following
claims.
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