U.S. patent application number 10/652468 was filed with the patent office on 2004-03-04 for hematopoietic differentiation of human embryonic stem cells.
Invention is credited to Kaufman, Dan S., Thomson, James A..
Application Number | 20040043484 10/652468 |
Document ID | / |
Family ID | 23728955 |
Filed Date | 2004-03-04 |
United States Patent
Application |
20040043484 |
Kind Code |
A1 |
Kaufman, Dan S. ; et
al. |
March 4, 2004 |
Hematopoietic differentiation of human embryonic stem cells
Abstract
Disclosed herein are methods of obtaining human hematopoietic
cells from human embryonic stem cells using mammalian stromal
cells. Hematopoietic cells derived in this way are useful for
creating cell cultures suitable for transplantation, transfusion,
and other purposes.
Inventors: |
Kaufman, Dan S.; (Madison,
WI) ; Thomson, James A.; (Madison, WI) |
Correspondence
Address: |
Nicholas J. Seay
Quarles & Brady LLP
P.O. Box 2113
Madison
WI
53701-2113
US
|
Family ID: |
23728955 |
Appl. No.: |
10/652468 |
Filed: |
August 29, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10652468 |
Aug 29, 2003 |
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09940175 |
Aug 27, 2001 |
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6613568 |
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09940175 |
Aug 27, 2001 |
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09435578 |
Nov 8, 1999 |
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6280718 |
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Current U.S.
Class: |
435/372 |
Current CPC
Class: |
C12N 2501/115 20130101;
C12N 2502/1394 20130101; C12N 5/0647 20130101; A61K 2035/124
20130101; C12N 2506/02 20130101; A61K 2035/122 20130101; C12N
2502/13 20130101 |
Class at
Publication: |
435/372 |
International
Class: |
C12N 005/08 |
Claims
We claim:
1. A method for obtaining human hematopoietic cells, comprising
exposing a human embryonic stem cell culture to mammalian
hematopoietic stromal cells so as to thereby create human
hematopoietic cells.
2. The method of claim 1, wherein at least some of the human
hematopoietic cells that are so created are CD34.sup.+.
3. The method of claim 1, wherein at least some of the human
hematopoietic cells that are so created are capable of forming
hematopoietic cell colony forming units in methylcellulose
culture.
4. The method of claim 1, wherein the stromal cells are selected
from the group consisting of bone marrow cells and embryonic yolk
cells.
5. A human hematopoietic cell culture which was derived from a
human embryonic stem cell culture in vitro.
6. The cell culture of claim 5, wherein the cell culture is capable
of forming hematopoietic cell colony forming units in
methylcellulose culture.
7. The cell culture of claim 5, wherein the cell culture has at
least some hematopoietic cells that are CD34.sup.+.
8. A method of transplanting human cellular material into a human
recipient host, comprising: obtaining human hematopoietic cells
which have been derived in vitro from an embryonic stem cell
culture; obtaining a selected human cellular material other than
hematopoietic cells, the selected non-hematopoietic material having
major histocompatibility complex compatibility to the hematopoietic
cells; and transplanting both the hematopoietic cells and selected
human non-hematopoietic cellular material into the human host.
9. The method of claim 8, wherein the selected human cellular
material is human pancreatic islets.
10. The method of claim 8, wherein the selected human cellular
material is human oligodendrocytes.
Description
CROSS REFERENCES TO RELATED APPLICATIONS
[0001] Not applicable.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
BACKGROUND OF THE INVENTION
[0002] The present invention relates to the use of human embryonic
stem cells to create blood-related cells, and the use of those
blood-related cells for various purposes.
[0003] Techniques for isolating stable cultures of human embryonic
stem cells have recently been described by our laboratory. See U.S.
Pat. No. 5,843,780 and J. Thomson et al., 282 Science 1145-1147
(1998). The disclosure of these publications and of all other
publications referred to herein are incorporated by reference as if
fully set forth below.
[0004] We have deposited two of our human embryonic stem cell lines
with the American Type Culture Collection, 10801 University
Boulevard, Manassas, Va. 20110-2209 U.S.A. on Jul. 7, 1999 and Jul.
15, 1999 respectively (with accession numbers PTA-313 and PTA-353
respectively). These deposits are under the conditions of the
Budapest Treaty. Taxonomic descriptions of these deposits are human
embryonic stem cell lines H1 and H9 respectively. It has been
proposed in these publications that such cell lines may be used
for, among other things, providing a source of specified cell lines
of various types for research, transplantation and other
purposes.
[0005] Under the storage and culturing conditions described in
these publications the cell lines are maintained long term without
differentiation into specific cell types. When the cell lines are
subsequently injected into immunodeficient mice, they form
teratomas demonstrating differentiation into multiple tissue
types.
[0006] When ES cells are used to produce desired cells, it is often
preferable to optimize differentiation towards specific cell types.
In the case of hematopoietic cells it is desirable that this result
in hematopoietic cells that can be isolated and used to form
multiple hematopoietic lineages. These cells may include, but not
be limited to, hematopoietic stem cells.
[0007] Hematopoietic stem cell populations have been isolated
directly from bone marrow. See C. Baum et al. 89 PNAS U.S.A.
2804-2808 (1992). However, this relies on a supply of bone marrow
to obtain the cells.
[0008] There have also been some attempts to direct murine
embryonic cell populations towards hematopoietic cells. See e.g.
U.S. Pat. No. 5,914,268; G. Keller, 7 Current Opinion In Cell
Biology, 862-869 (1995); and T. Nakano et al. 265 Science 1098-1101
(1994). See also M. Weiss, 11 Aplastic Anemia And Stem Cell
Biology, 1185-1195 (1997); and S. Morrison et al., 11 Annu. Rev.
Cell Dev. Biol., 35-71 (1995).
[0009] However, applying these teachings to primates has proven
difficult. For example, in F. Li et al., 92 Blood 368a (1998) there
was a discussion of techniques for differentiation of rhesus
embryonic stem cell lines using a stromal cell line and exogenous
cytokines. However, that group has more recently reported that
their techniques had inadequate formation of colonies.
[0010] The treatment of various diseases by tissue transplantation
has become routine. However, there can be waiting lists to obtain
natural donated organs, cells, or tissue. Even when the natural
donor material becomes available there is often a problem with
rejection. Traditional approaches for suppressing an immune
response of recipients have drawbacks. For example,
immunosuppressive drugs are costly and often have side effects.
[0011] In WO 98/07841 there was discussed techniques of deriving
embryonic stem cells that are MHC compatible with a selected donor
(e.g. transplanting a donor nucleus into an enucleated oocyte,
followed by derivation of the stem cells therefrom). The
application suggested that the resulting cells could be used to
obtain MHC compatible hematopoietic stem cells for use in medical
treatments requiring bone marrow transplantation.
[0012] However, some diseases such as type 1 diabetes mellitus or
multiple sclerosis involve an autoimmune response. For example,
merely transplanting pancreatic islets (which are MHC compatible to
the diseased individual) to replace destroyed pancreatic islets
will not provide sufficient long term reduction in type 1 diabetes
mellitus, as the immune system of the host will still attack the
transplanted islets.
[0013] It can therefore be seen that a need exists for techniques
for causing human embryonic stem cell cultures to differentiate to
desired hematopoietic colonies. Further, it is desired to develop
improved uses for hematopoietic cells.
BRIEF SUMMARY OF THE INVENTION
[0014] In one aspect the present invention provides a method for
obtaining human hematopoietic cells. One exposes a human embryonic
stem cell culture to mammalian hematopoietic stromal cells so as to
thereby create human hematopoietic cells. At least some of the
human hematopoietic cells that are so created are CD34.sup.+ and/or
are capable of forming hematopoietic cell colony forming units in
methylcellulose culture.
[0015] CD34 is a standard marker for hematopoietic stem cells, as
described in C. Baum et al. 89 PNAS U.S.A. 2804-2808 (1992) and S.
Morrison et al., 11 Annu. Rev. Cell Dev. Biol., 35-71 (1995). The
property of capability of forming a colony forming unit is
indicative that the cells have the desired characteristics to form
more differentiated hematopoietic lineages.
[0016] The stromal cells are preferably derived from bone marrow
cells or embryonic yolk sac cells. Murine stromal cells may be used
for this purpose. However, primate stromal and other mammalian
stromal cells should be suitable as well.
[0017] In another aspect the invention provides a human
hematopoietic cell which was derived from a human embryonic stem
cell culture in vitro, and is capable of forming hematopoietic cell
colony forming units in methylcellulose culture. As used in this
patent, the term "derived" is intended to mean obtained directly or
indirectly (e.g. through one or more intermediates or
passages).
[0018] In yet another aspect the invention provides a method of
transplanting human cellular material into a human recipient host.
One obtains human hematopoietic -cells which have been derived in
vitro from an embryonic stem cell culture. One then obtains a
selected human cellular material other than hematopoietic cells,
the selected non-hematopoietic material having major
histocompatibility complex compatibility to the hematopoietic
cells. One then transplants both the hematopoietic-cells and
selected human non-hematopoietic cellular material into the human
host.
[0019] For example, one can obtain human hematopoietic cells which
have been derived in vitro from an embryonic stem cell culture
(e.g. using the techniques described below). One also obtains human
pancreatic islets which have MHC compatibility to the hematopoietic
cells. Both the hematopoietic cells and pancreatic islets are then
transplanted into the human (preferably after the recipient's own
bone marrow has been inactivated).
[0020] The pancreatic islets can be obtained directly from a donor
whose cells were used to create the embryonic stem cell culture.
Alternatively, a single embryonic stem cell culture can be
differentiated along two different paths. In one process the above
technique can be used to create hematopoietic stem cells. These
cells should develop into multiple hematopoietic lineages when
transplanted into appropriate hosts. These lineages should include
lymphocytes which would be tolerant of other cells derived from the
same parental embryonic stem cells. In another process the stem
cells would be directed towards pancreatic islets.
[0021] In another example one could supply oligodendrocytes to a
human who has a multiple sclerosis condition. One obtains human
hematopoietic cells which have been derived in vitro from an
embryonic stem cell culture (e.g. using a technique described
below). One also obtains human oligodendrocytes which have MHC
compatibility to the bone marrow cells and transplants both the
bone marrow cells and oligodendrocytes into the human.
[0022] The same human whose genetic material was used to create the
embryonic stem cell can be a donor for the oligodendrocytes.
Alternatively, the same embryonic stem cell culture can be
differentiated along two separate paths to provide the two
transplantable materials.
[0023] With respect to either disease (and potentially other
autoimmune diseases) the immune-and auto-immune rejection problems
should be reduced by this technique. In this regard, the
recipient's original bone marrow can be totally or partially
inactivated by radiation or chemical means before the
transplantation. Thereafter, it is replaced at least in part by the
transplanted hematopoietic cells. The elimination/reduction of the
original bone marrow reduces the body's ability to create an
autoimmune response. The matching of the MHC of the replacement
bone marrow and the second transplantable material insures that the
second material won't be rejected by the transplanted bone
marrow.
[0024] Moreover, co-transplantation of hematopoietic cells and
other tissue can be done to promote acceptance of the second tissue
(e.g. heart muscle plus hematopoietic cells for treating heart
disease; hepatocytes plus hematopoietic cells for treating liver
disease). By creating hematopoietic chimeras improved acceptance of
tissues with similarly matched MHC type can be obtained.
[0025] The present invention should be suitable to obtain a wide
variety of hematopoietic cells of interest, such as erythroid
cells, granulocyte cells, macrophages, lymphocyte precursors,
monocytes, B cells, T cells, and the like. In this regard, colonies
of differentiated ES cells develop into hematopoietic colonies when
harvested, separated into single cells, and plated into appropriate
cultures. These colonies demonstrate the development of
colony-forming cells which proliferate into colony-forming units
(including colony forming unit-erythroid (CFU-E), blast forming
unit-eythroid (BFU-E), colony forming unit-macrophage (CFU-M),
colony forming unit-granulocyte/macrophage (CFU-GM) and colony
forming unithigh proliferative potential (CFU-HPP)). The
identification of colony forming cells indicates the
differentiation of embryonic stem cells into hematopoietic cells
capable of expanding into defined hematopoietic lineages under
defined conditions.
[0026] The objects of the present invention therefore include
providing:
[0027] (a) methods of the above kind for obtaining hematopoietic
cells;
[0028] (b) cells derived using those methods; and
[0029] (c) methods for using those derived cells for
transplantation, transfusion and other purposes. These and still
other objects and advantages of the present invention will be
apparent from the description of the preferred embodiments that
follows. However, the claims should be looked to in order to judge
the full scope of the invention.
DETAILED DESCRIPTION
Embryonic Stem Cell Culture
[0030] The previously described human ES cell line Hi was used for
the majority of experiments, albeit some of the following studies
were done with the previously described ES cell lines H9 (or H9.2)
with similar results. These cells were removed from frozen (liquid
nitrogen) stocks of cells derived from the original isolated and
propagated cell line. The Hi ES cells were grown in 6 well culture
dishes (Nunclon, Fisher).
[0031] The dish was first coated with 0.1% gelatin solution (Sigma)
for one or more days in a 37.degree. C./5% CO.sub.2 incubator.
After the one or more days, the gelatin solution was removed and
the wells of the plate were next coated with irradiated mouse
embryonic fibroblast (MEF) cells. MEF cells were derived from day
12-13 mouse embryos in medium consisting of DMEM (GibcoBRL)
supplemented with 10% fetal bovine serum (Hyclone or Harlan), 2 mM
1-glutamine (GibcoBRL), and 100 units/ml. Penicillin, 100 mg/ml
streptomycin (Sigma).
[0032] The MEF cells were irradiated with 5500 cGy from a cesium
source prior to plating in the wells. The MEFs were added at a
density of 5.times.10.sup.4cells/ml, 2.5 ml/well. The plate coated
with MEFs was then placed in 37.degree. C./5% CO.sub.2 incubator
for one or more days until addition of ES cells.
[0033] ES cells were passed onto new MEFs at approximately 5-8 day
intervals. The time depends on cell density and morphologic
appearance of differentiation. For passage, the medium in a well of
ES cells was removed and 1-2 ml of medium containing 1 mg/ml
collagenase IV in DMEM (GibcoBRL) was added. The plate was then
placed at 37.degree. C./5% CO.sub.2 for 5-20 minutes until the
colonies of ES cells began to round up.
[0034] The well was then scraped with a 5 ml pipette to detach the
ES cells from the plate. The contents of the harvested well were
placed in a 15 ml conical tube (Fisher) and spun in a centrifuge at
1000 rpm for 5 minutes. The medium was removed and 10 ml of fresh
medium was added. This ES cell medium consists of F12/DMEM
(GibcoBRL)) supplemented with 20% serum replacement medium
(GibcoBRL), 8 ng/ml of bFGF (GibcoBRL), 1% nonessential amino acid
solution (GibcoBRL), 1 mM 1 glutamine (GibcoBRL), and 0.1M
.beta.-mercaptoethanol.
[0035] The cells were again spun (5 min/1000 rpm), medium removed
and resuspended at a concentration of 2.5 ml of medium for each
(typically 15 ml medium for plating into 6 new wells, this would be
a 1:6 passage). The cells were then pipetted into the wells of a
plate that had been previously coated with MEFs as described above.
The cells were evenly distributed into each well and the plate was
placed in an incubator at 37.degree. C./5% CO.sub.2.
[0036] At times if there were colonies of ES cells showing
morphologic appearance of differentiation prior to cell passage,
these colonies were removed by gentle scraping with a pulled glass
pipette. This was done with observation through a dissecting
microscope. After removal of the differentiated cells, the
remaining colonies were passaged as above.
[0037] After passage, each well of ES cells was "fed" with fresh
medium at 24-48 hour intervals. Here, the medium of each well was
removed and 2.5 ml of fresh ES medium was added. All feeding and
passage of ES cells were done in a sterile environment.
Differentiation of ES Cells
[0038] To promote hematopoietic differentiation of the human ES
cells, the ES cells were harvested as above. The cells were then
plated in 6 well plates coated with a mammalian stromal cell. In
one experiment we used C166 cells that were previously irradiated
with 2500 cGy. The C166 cells were originally obtained from the
yolk sac of mice at embryonic day 12 and were graciously provided
by Dr. Robert Auerbach (UW-Madison).
[0039] In another experiment, S17 cells were used. They were
originally obtained from mouse bone marrow, and were graciously
provided by Dr. Kenneth Dorshkind (then at UCRiverside, now at
UCLA).
[0040] The C166 or S17 cells were plated at a density of
1.times.10.sup.5 cells/ml, 2.5 ml/well. The ES cells plated onto
either S17 of C166 cells were then allowed to grow in a medium
consisting of DMEM (GibcoBRL) supplemented with 20% fetal bovine
serum (Hyclone), 1% nonessential amino acid solution, 0.1 M
.beta.-mercaptoethanol, and 1 mM 1 glutamine. This medium was
replaced in each well at 24-72 hour intervals with fresh medium. In
selecting an appropriate medium, one merely needs to provide
conventional conditions for cell growth, albeit supplemented with
the specified stromal cells.
[0041] After 3-7 days from plating onto S17 or C166 cells, the ES
cells began to visually appear differentiated in that they did not
have the same uniform appearance as the undifferentiated ES cells
maintained on MEF feeder cells. The colonies of ES cells began to
form multiple different cell types. Some of these colonies had
regions that appeared to consist of cells with a cobblestone
morphology indicative of colonies of early hematopoietic progenitor
cells.
Confirming Blood-Related Cells
[0042] One method to determine the presence of appropriate
hematopoietic cells is to assay for hematopoietic colony forming
cells (CFCs) in semisolid methylcellulose containing medium. Here,
the ES cells were allowed to differentiate on either C166 or S17
cells for 2-3 weeks, maintained as described above. After this time
the medium was removed. 2.5 ml of calcium and magnesium free
phosphate buffered saline (PBS) was added for 2-5 minutes, removed,
and 1.5 ml. of trypsin (0.125%)-EDTA (1mM) medium was added.
[0043] The cells were then placed at 37.degree. C./5% CO.sub.2 for
10 minutes. After this time, the colonies began to disassociate.
The cells were further disassociated by pipetting and scraping the
wells. The cells were placed in a 15 ml. conical, spun 5 min/1000
rpm, medium removed and 10 ml fresh medium (DMEM+10%
FBS+1-glutamine+pen/strep) was added, and spun again. The cells
were then suspended in 5 ml medium and passaged through a 100 mM
nytex filter to remove clumps of cells.
[0044] The filter was washed with an additional 5 ml medium. The
disassociated/filtered cells were then counted on a hemacytometer
and 1.times.10.sup.6 (usually, but not always this many cells)
cells were placed in a new 15 ml conical. These cells were then
spun, medium removed and 5 ml medium consisting of IMDM (GibcoBRL)
supplemented with 2% fetal bovine serum (Hyclone) was added. Cells
were spun, medium removed and 250 ul medium (IMDM+2% FBS) was
added.
[0045] In accordance with the specified test conditions, these
cells were then added to 2.5 ml of Methocult GF+ H4435 medium
(StemCell Technologies). This medium consists of 1.0%
methylcellulose, supplemented with 30% FBS, 20 ng/ml IL-3, 20 ng/ml
IL-6, 50 ng/ml stem cell factor, 3 units/ml erythropoietin, 20
ng/ml GM-CSF, 20 ng/ml G-CSF, 2 mM 1-glutamine, 0.1 mM
b-mercaptoethanol, 1% bovine serum albumin. The cells in
methylcellulose were then vortexed vigorously and then 1.1 ml of
the mixture was plated onto a P35 plastic dish (Stem Cell
Technologies), spread evenly on the dish and placed at 37.degree.
C./5% CO.sub.2.
[0046] Duplicate plates of each sample were typically plated with
4.times.10.sup.5 cells/plate. After 14-21 days, the plates were
analyzed under a microscope for the presence of hematopoietic
colonies. The colonies were identified by comparison to a colony
atlas (StemCell Technologies) or the book: Culture of Hematopoietic
Cells, RI Freshney, IB Pragnell, MG Freshney, eds., Wiley-Liss,
Inc. 1994. Colonies were identified as one of the following: colony
forming unit-erythroid (CFU-E), blast forming uniteythroid (BFU-E),
colony forming unit-macrophage (CFU-M), colony forming
unit-granulocyte/macrophage (CFU-GM) or colony forming unit-high
proliferative potential (CFUHPP).
[0047] The presence of the desired hematopoietic cells can also be
confirmed by flow cytometry. One can look for specified cell
surface antigens by flow cytometry. Here, ES cells differentiated
on S17 cells or C166 cells as described above for 14-21 days, were
harvested with trypsin/EDTA as described above and passed through a
100 mM nytex filter. The filtered cells were counted on a
hemacytometer, then aliquotted into 15.times.75 plastic tubes
(Fisher) at approximately 1.times.10.sup.5 cells/tube. The cells
were then spun, medium removed and 2-3 ml of FACS medium was added.
(FACS medium is PBS with 0.5% BSA (Sigma), 0.1% sodium azide
(Sigma)).
[0048] The cells were again spun and medium removed. Next an
antibody directly linked to a fluorescent marker (FITC or PE) was
added to the wells at a concentration as recommended by the
supplier. Cells have been analyzed with the following antibodies:
CD34-FITC (Immunotech), CD45-PE (Pharmingen). IgG1-FITC and IgG1-PE
were used as isotype controls for non-specific staining of the
cells. Cells were incubated with the appropriate antibody for
approximately 30 min on ice, washed 1-2 times with 2-3 ml FACS
medium and resuspended in approximately 0.5 ml FACS medium.
[0049] The antibody labeled cells were then analyzed using a
FACScan (Becton Dickinson) as per manufacturers recommendations.
The presence of dead cells was determined by addition of propidium
iodide (1 mg/ml solution, 5 ul added per tube) or 7-AAD
(Calbiochem) (0.2 mg/ml ,5 ul/tube). The software for analysis was
either PC Lysis or Cellquest.
[0050] The following experimental techniques were used to analyze
antigen expression by immunohistochemistry (IHC). Here,
differentiated ES cells that have been co-cultured with either C166
or S17 as above, were harvested with trypsin/EDTA as above. The
cells were resuspended in medium containing DMEM supplemented with
10% FBS at a concentration of approximately
1.times.10.sup.4-1.times.10.sup.5. "Cytospin" preparations of these
cells were then made by spinning 1.times.10.sup.3-1.times.10.sup-
.4 cells onto a glass slide (Superfrost/plus, Fisher) with a
Cytospin II centrifuge (Shanndon).
[0051] These slides were then fixed with cold acetone and stored
frozen at -20.degree. C. For IHC staining the slides were thawed at
room temperature and the cell pellet was outlined with a wax pen
(DAKO). The cells were then stained as follows using a Vectastain
ABC kit (Vector Laboratories, Burlingame, CA), all incubations were
at room temperature. 100-200 ul PBS was added onto the cells for 5
minutes then removed. Vectastain blocking antibody solution (horse
serum) was then added onto the cells for 15 minutes. The cells were
then blotted dry and 100-200 ul of primary antibody solution was
added. The primary antibodies were: IgG1 (1 ug/sample, Sigma),
anti-CD34 (0.5 ug/sample, Immunotech), anti-CD45 (1 ug/sample,
DAKO), anti-class I (1 ug/sample, gift from Dr. Paul Leibson, Mayo
Clinic), anti-CD14 (1 ug/sample, Pharmingen), anti-CD31 (1
ug/sample, Pharmingen).
[0052] Primary antibody was added for 30 minutes followed by PBS
for 10 minutes. Next, biotinylated anti-IgG antibody was added
(Vectastain kit, solution B) for 30 minutes followed by PBS for 10
minutes. Next Vectastain ABC solution was added for 30 minutes at
room temperature followed by PBS for 10 minutes. Next DAB solution
(Vectastain) was added for 5 minutes followed by washing under
running tap water for 10 minutes. In some experiments, the slides
were then counterstained with Gill's hematoxylline solution (Vector
labs) for 3 minutes followed by washing with running tap water for
10 minutes. The slides were then air dried. Cells staining positive
appear brown.
[0053] CD34.sup.+was demonstrated within a mixed population of
cells (about 1%) after 2-3 weeks. Even more importantly,
differentiated ES cells were shown to develop into hematopoietic
colonies when harvested, separated into cells and plated into
methylcellulose (semi-solid) cultures.
Transplantation
[0054] Currently hematopoietic cell transplantation is conducted
clinically primarily for patients who have received high dose
chemotherapy for treatment of malignancies. These patients
typically receive a heterogeneous mixture of hematopoietic cells
either from an autologous or allogeneic source. Human ES-derived
hematopoietic stem cells will at minimum provide a more homogeneous
cell population for hematopoietic cell transplantation.
[0055] Further, as discussed above, the MHC characteristics of the
transplantation can now be controlled, thereby enabling treatment
of autoimmune diseases. For example, both hematopoietic stem cells
(HSCs) and a second lineage (e.g. pancreatic islets for diabetes or
oligodendrocytes for multiple sclerosis) could be derived from the
same parental ES cell line. With both lineages available, a
hematopoietic chimera could be first created by performing a fully
allogeneic hematopoietic cell transplant (HCT). The established
state of chimerism would allow the recipient's immune system to
"see" the subsequent transplant of the second cell type (e.g.
pancreatic islets cell or oligodendrocyte) as "self" and should not
be rejected.
[0056] Note for example that oligodendrocytes have been obtained
from mouse ES cells (0. Brustle et al., 285 Science 754-6 (1999)),
as have cardiac muscle cells (M. Klug et al., 98 J. Clin. Invest.
216-224 (1996)).
[0057] This method of creating hematopoietic chimeras will also
promote acceptance of tissues transplanted for reasons other than
autoimmunity. In this regard, mice receiving allogeneic
hematopoietic stem cells do not reject other tissues with the same
genetic background as the hematopoietic cells, but will still
reject thirdparty grafts. See K. Gandy et al., 65 Transplantation
295-304 (1998).
[0058] In addition to animal studies, there are now clinical case
reports of human patients who have previously received a
hematopoietic cell transplant later requiring a solid organ
(kidney) transplant. In these instances, the kidney transplant from
the same person who had previously supplied the bone marrow
transplant is immunologically accepted without further
immunosuppression. See T. Spitzer et al., 68 Transplantation
480-484 (1999).
[0059] Work in canine models and more recently in human clinical
trials has shown that milder non-myeloablative conditioning
regimens can be used to better prepare hosts for allogenic HCT.
Here, only moderate doses of total body irradiation and a short
course of immunosuppression are used to prepare the hosts prior to
receiving allogeneic HCTs.
[0060] Even though the preferred embodiments have been described
above, it will be appreciated by those skilled in the art that
other modifications can be made within the scope of the invention.
For example, while two specific stromal type cells have been
selected for use, many others are also suitable. For example, one
publicly available stromal cell line is the M2-10B4 cell line
having ATCC designation number CRL-1972.
[0061] Further, while the above description focuses on the creation
of precursors for red blood cells and bone marrow, various other
blood-related cells of interest can be obtained in quantity using
the above techniques. See also U.S. Pat. No. 5,914,268. Thus, the
claims should be looked to in order to judge the full scope of the
invention.
Industrial Applicability
[0062] The invention provides blood-related cells useful for
transplantation, research and other purposes.
* * * * *