NZ714064B2 - Improving organs for transplantation - Google Patents
Improving organs for transplantation Download PDFInfo
- Publication number
- NZ714064B2 NZ714064B2 NZ714064A NZ71406414A NZ714064B2 NZ 714064 B2 NZ714064 B2 NZ 714064B2 NZ 714064 A NZ714064 A NZ 714064A NZ 71406414 A NZ71406414 A NZ 71406414A NZ 714064 B2 NZ714064 B2 NZ 714064B2
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- cells
- stem cells
- organ
- cell
- tissue
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/70—Undefined extracts
- C12N2500/80—Undefined extracts from animals
- C12N2500/84—Undefined extracts from animals from mammals
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/03—Coculture with; Conditioned medium produced by non-embryonic pluripotent stem cells
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0018—Culture media for cell or tissue culture
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0607—Non-embryonic pluripotent stem cells, e.g. MASC
Abstract
The invention provides methods and compositions that improve the success of organ or tissue transplantation. The methods and compositions are directed to exposing a desired organ or tissue to stem cells prior to transplantation. In a particular embodiment these stem cells are multipotent adult progenitor stem cells (MAPCs or MASCs). In one embodiment, the stem cells reduce the deleterious effects of ischemia on an organ or tissue designated to be harvested for transplantation or that has been harvested for transplantation. In another embodiment in which an organ or tissue designated for transplantation is perfused ex vivo, the method involves reducing ischemic reperfusion injury by perfusing the organ or tissue with a medium that contains stem cells. nitor stem cells (MAPCs or MASCs). In one embodiment, the stem cells reduce the deleterious effects of ischemia on an organ or tissue designated to be harvested for transplantation or that has been harvested for transplantation. In another embodiment in which an organ or tissue designated for transplantation is perfused ex vivo, the method involves reducing ischemic reperfusion injury by perfusing the organ or tissue with a medium that contains stem cells.
Description
IMPROVING ORGANS FOR TRANSPLANTATION
SEQUENCE LISTING
The instant application contains a sequence listing which has been submitted electronically in
ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on April
8, 2014, is named ATH-022234USORD_SL.txt and is 7,517 bytes in size.
FIELD OF THE INVENTION
The field of the invention is organ transplantation and providing methods and compositions that
improve the success of organ transplantation. The methods and compositions are directed to exposing a
desired organ, prior to or during transplantation, to stem cells. In one embodiment, the stem cells reduce
the deleterious effects of ischemia on an organ designated to be harvested for transplantation or that has
been harvested for transplantation. In another embodiment, the organ is a lung. In a further embodiment
in which an organ designated for transplantation is perfused ex vivo, the method involves reducing
ischemic reperfusion injury by perfusing the organ with a medium that contains stem cells.
BACKGROUND OF THE INVENTION
Organ transplantation represents the preparation and harvesting of an organ from a donor or a
donor site (if the donor and recipient are the same), and the implantation, maintenance and/or use of the
organ into or by the recipient of the donated organ. It has been estimated that there are more than 50,000
organ transplants performed per year in the major healthcare markets (e.g., U.S., Europe and Japan), and
that there are more than 170,000 patients on waiting lists for organ transplants. Demand for healthy
organs significantly outstrips the supply.
A major challenge in organ transplantation has been transplant rejection, which can lead to
significant complications in organ function or to transplant failure. In general, this has been addressed
through the matching of donors and recipients who have highly similar serotypes, and through the use of
immunosuppressive drugs to manage the immunological response underlying transplant rejection.
Another major challenge has been preservation of organ viability prior to and during the
implantation procedure. The removal, storage and transplantation of an organ may profoundly affect the
internal structure and function of the organ and can influence significantly the degree to which the return
of normal organ function is delayed or prevented after transplantation is completed. Such organ injury
occurs primarily as a result of ischemia and hypothermia, but may also be related to reperfusion of the
organ ex vivo or during implantation. Techniques for organ preservation, including ex vivo perfusion,
serve to minimize this damage to promote optimal graft survival and function. But, even with these
techniques, the organ health will decline is many cases, affecting transplantation outcome, and in some
cases, the decline is so significant that the donated organs are rejected prior to transplantation as non-
viable.
A technology that addresses these important challenges in organ transplantation should have a
substantial impact on patient quality of life and survival, and on the treatment of the complications
associated with transplantation.
SUMMARY OF THE INVENTION
The invention provides a method of treating an organ for transplantation, comprising exposing the organ
to exogenous stem cells, wherein the stem cells are non-embryonic, non-germ cells that express
telomerase, have a normal karyotype, are not tumorigenic, and wherein the stem cells have undergone at
least 10-40 cell doublings in cell culture prior to their exposure to the organ, wherein the organ is exposed
to the stem cells prior to transplantation into a recipient.
The invention further provides a method comprising transplanting an organ that has been
exposed to exogenous stem cells prior to, during, and/or after transplantation. Exposure to the stem cells
can improve the probability of a successful organ transplantation. Accordingly, the invention is directed
to the following embodiments.
In one embodiment, the method may involve tolerizing the organ by contacting the organ with
exogenous stem cells prior to, during, and/or after transplantation. By tolerizing the organ, that organ is
better prepared to be accepted by a recipient without significant immunological interference. Tolerization
can be achieved, for example, by the induction of T-regulatory cells in the organ (see, e.g., Eggenhofer et
al., Stem Cells Translation Medicine 2013;2:000-000).
In one embodiment, the invention is directed to a method to reduce injury in an organ ex vivo by
contacting the organ with a medium that contains exogenous stem cells prior to, during, and/or after
transplantation.
In one embodiment, the injury occurs as a result of ischemic reperfusion injury.
In one embodiment, the method is directed to reducing general tissue or cell degradation in the
organ to be transplanted. This may result from factors including, but not limited to, ischemia,
hypothermia and reperfusion. Accordingly, in one embodiment, the invention is directed to reducing
injury as a result of one or more of these events. Such events may be caused, at least in part, by one or a
combination of the following: (1) immunomodulation of TH1 T-cells to TH2 T-cells; (2)
immunomodulation of M1 macrophages to M2 macrophages (e.g., causing a shift from a pro-
inflammatory response to an anti-inflammatory response); (3) inhibiting the infiltration of neutrophils
(e.g., by reducing the cell surface receptors); (4) shifting neutrophils from being pro-inflammatory to anti-
inflammatory; and (5) cytoprotection or anti-apoptotic effects generated by the exogenous stem cells.
The events described herein may result in inflammation, other immunological response, cytokine
production, cell apoptosis, and other events that affect the viability of an organ and suitability for
transplantation. Accordingly, in one embodiment, the invention is directed to reducing the deleterious
effects of these events by administering exogenous stem cells to an organ that is subject to these events or
in which these events have already occurred.
Examples of events that may result in inflammation, other immunological response, cytokine
production, cell apoptosis, and other events that affect the viability of an organ and suitability for
transplantation can include, but are not limited to, those associated with endothelial response, reactive
oxygen species, complement, and leukocytes. Events associated with endothelial response can include,
but are not limited to, expression of certain pro-inflammatory gene products (e.g., leukocyte adhesion
molecules, cytokines) and/or bioactive agents (e.g., endothelin, thromboxane A ) and/or repression of
other “protective” gene products (e.g., constitutive nitric oxide synthase, thrombomodulin) and/or
bioactive agents (e.g., prostacyclin, nitric oxide). Events associated with reactive oxygen species (e.g.,
(O2–), (OH–), (HOCl), (H2O2), and nitric oxide-derived peroxynitrite) can include, but are not limited to,
direct damage to cellular membranes by lipid peroxidation, stimulating leukocyte activation and
chemotaxis by activating plasma membrane phospholipase A2 to form arachidonic acid (thromboxane A2
and leukotriene B4), and/or increasing leukocyte activation, chemotaxis, and leukocyte-endothelial
adherence after ischemic reperfusion. Events associated with complement activation, such as C3a, C5a,
iC3b, C5b9 (C5a is most potent) can include, but are not limited to, formation of several pro-
inflammatory mediators that alter vascular homeostasis by, e.g., compromising blood flow to an ischemic
organ by altering vascular homeostasis and increasing leukocyte–endothelial adherence. Events
associated with leukocytes can include, but are not limited to, leukocyte activation, chemotaxis,
leukocyte–endothelial cell adhesion and transmigration, which may further lead to mechanical
obstruction, as leukocytes release toxic ROS, proteases, and elastases, resulting in increased
microvascular permeability, edema, thrombosis, and parenchymal cell death.
In one embodiment, the organ is selected from the group including, but not limited to, lung,
kidney, heart, liver, pancreas, thymus, gastrointestinal tract and composite allografts, such as limbs, faces
and the like, and tissues including, but not limited to, cornea, skin, veins, arteries, bones, tendons and
valves, such as heart valves and the like.
In one embodiment, the stem cells reduce inflammation in the organ. For example, the organ
can be exposed to the stem cells for a time and dose sufficient to reduce inflammation in the organ.
In one embodiment, the stem cells reduce the occurrence of inflammatory cells in the organ. For
example, the organ can be exposed to the stem cells for a time and with a dose sufficient to reduce the
occurrence of inflammatory cells in the organ.
In one embodiment, the stem cells reduce inflammatory cytokines in the organ. For example,
the organ can be exposed to the stem cells for a time and with a dose sufficient to reduce inflammatory
cytokines in the organ.
In one embodiment, the stem cells reduce the occurrence of pulmonary edema. For example, the
organ can be exposed to the stem cells for a time and with a dose sufficient to reduce the occurrence of
pulmonary edema.
In one embodiment, the stem cells increase the occurrence of IL-10 expression (protein and/or
mRNA) in pulmonary tissue. For example, the organ can be exposed to the stem cells for a time and with
a dose sufficient to increase the occurrence of IL-10 expression in pulmonary tissue.
In one embodiment injury results from hypoxia in the organ.
In one embodiment, the stem cells reduce the effects of hypoxia in the organ.
In one embodiment, the stem cells are administered at any time between removal of the organ
from the donor and transplantation into the recipient.
In one embodiment, the stem cells are exposed to the organ during the transplantation procedure.
In one embodiment, the organ can be exposed to the stem cells while the organ is still intact in
the donor but before removal of the organ from the donor.
In one embodiment, the organ can be exposed to the stem cells for a period of time. The period
of time can depend upon the particular organ. For example, the period of time can be about 1-2 hours,
about 2-3 hours, about 3-4 hours, about 4-5 hours, about 5-6 hours, about 7-8 hours, about 8-9 hours,
about 9-10 hours, or about 10 hours or more. One example of a suitable period of time is described by
Zhao et al., BMC Medicine 2012, 10:3, which is incorporated by reference herein for the teaching of an ex
vivo procedure, including suitable time periods, for an intravenous ex vivo cell process.
In one embodiment, the concentration of stem cells exposed to the organ can depend upon the
particular organ. For example, the concentration of cells exposed to the organ can be about 0.01 to about
7 5 7 6
x10 cells/ml, about 1 x 10 cells/ml to about 5 x10 cells/ml, or about 10 x 10 cells/ml.
In another embodiment, the concentration of stem cells exposed to the organ can be about 1 x
5 5 6
cells/kg organ to about 5 x 10 cells/kg organ, about 5 x 10 cells/kg organ to 1 x 10 cells/kg organ to
6 6 7 7
x 10 cells/kg organ, about 5 x 10 cells/kg organ to 1 x 10 cells/kg organ, about 1 x 10 cells/kg organ
7 7 8
to 1.5 x 10 cells/kg organ, or about 1 x 10 cells/kg organ to 2 x 10 cells/kg organ.
In other embodiments, the stem cells are contained in a fluid for perfusion into the organ or in a
carrier for intra-organ (such as intra-bronchially) administration.
In another embodiment, the stem cells are contained in a medium in which the organ is contacted
prior to transplantation, such as a medium in which the organ is bathed rather than being perfused.
The inventors contemplate using any desired stem cell in the methods of the invention. These
include, but are not limited to, embryonic stem cells, non-embryonic multipotent stem cells, mesenchymal
stem cells, neural stem cells, induced pluripotent stem cells, and the like. In one embodiment, the stem
cells can be non-HLA matched, allogeneic cells.
Cells include, but are not limited to, cells that are not embryonic stem cells and not germ cells,
having some characteristics of embryonic stem cells, but being derived from non-embryonic tissue, and
providing the effects described in this application. The cells may naturally achieve these effects (i.e., not
genetically or pharmaceutically modified). However, natural expressors can be genetically or
pharmaceutically modified to increase potency.
The cells may express pluripotency markers, such as oct4. They may also express markers
associated with extended replicative capacity, such as telomerase. Other characteristics of pluripotency
can include the ability to differentiate into cell types of more than one germ layer, such as two or three of
ectodermal, endodermal, and mesodermal embryonic germ layers. Such cells may or may not be
immortalized or transformed in culture. The cells may be highly expanded without being transformed and
also maintain a normal karyotype. In one embodiment, the non-embryonic stem, non-germ cells may
have undergone a desired number of cell doublings in culture. For example, non-embryonic stem, non-
germ cells may have undergone at least 10-40 cell doublings in culture, such as 30-35 cell doublings,
wherein the cells are not transformed and have a normal karyotype. The cells may differentiate into at
least one cell type of each of two of the endodermal, ectodermal, and mesodermal embryonic lineages and
may include differentiation into all three. Further, the cells may not be tumorigenic, such as not
producing teratomas. If cells are transformed or tumorigenic, and it is desirable to use them for infusion,
such cells may be disabled so they cannot form tumors in vivo, as by treatment that prevents cell
proliferation into tumors. Such treatments are well known in the art.
Cells include, but are not limited to, the following numbered embodiments:
1. Isolated expanded non-embryonic stem, non-germ cells, the cells having undergone at least
-40 cell doublings in culture, wherein the cells express oct4, are not transformed, and have a normal
karyotype.
2. The non-embryonic stem, non-germ cells of 1 above that further express one or more of
telomerase, rex-1, rox-1, or sox-2.
3. The non-embryonic stem, non-germ cells of 1 above that can differentiate into at least one
cell type of at least two of the endodermal, ectodermal, and mesodermal embryonic lineages.
4. The non-embryonic stem, non-germ cells of 3 above that further express one or more of
telomerase, rex-1, rox-1, or sox-2.
5. The non-embryonic stem, non-germ cells of 3 above that can differentiate into at least one
cell type of each of the endodermal, ectodermal, and mesodermal embryonic lineages.
6. The non-embryonic stem, non-germ cells of 5 above that further express one or more of
telomerase, rex-1, rox-1, or sox-2.
7. Isolated expanded non-embryonic stem, non-germ cells that are obtained by culture of non-
embryonic, non-germ tissue, the cells having undergone at least 40 cell doublings in culture, wherein the
cells are not transformed and have a normal karyotype.
8. The non-embryonic stem, non-germ cells of 7 above that express one or more of oct4,
telomerase, rex-1, rox-1, or sox-2.
9. The non-embryonic stem, non-germ cells of 7 above that can differentiate into at least one
cell type of at least two of the endodermal, ectodermal, and mesodermal embryonic lineages.
10. The non-embryonic stem, non-germ cells of 9 above that express one or more of oct4,
telomerase, rex-1, rox-1, or sox-2.
11. The non-embryonic stem, non-germ cells of 9 above that can differentiate into at least one
cell type of each of the endodermal, ectodermal, and mesodermal embryonic lineages.
12. The non-embryonic stem, non-germ cells of 11 above that express one or more of oct4,
telomerase, rex-1, rox-1, or sox-2.
13. Isolated expanded non-embryonic stem, non-germ cells, the cells having undergone at least
-40 cell doublings in culture, wherein the cells express telomerase, are not transformed, and have a
normal karyotype.
14. The non-embryonic stem, non-germ cells of 13 above that further express one or more of
oct4, rex-1, rox-1, or sox-2.
15. The non-embryonic stem, non-germ cells of 13 above that can differentiate into at least one
cell type of at least two of the endodermal, ectodermal, and mesodermal embryonic lineages.
16. The non-embryonic stem, non-germ cells of 15 above that further express one or more of
oct4, rex-1, rox-1, or sox-2.
17. The non-embryonic stem, non-germ cells of 15 above that can differentiate into at least one
cell type of each of the endodermal, ectodermal, and mesodermal embryonic lineages.
18. The non-embryonic stem, non-germ cells of 17 above that further express one or more of
oct4, rex-1, rox-1, or sox-2.
19. Isolated expanded non-embryonic stem, non-germ cells that can differentiate into at least
one cell type of at least two of the endodermal, ectodermal, and mesodermal embryonic lineages, said
cells having undergone at least 10-40 cell doublings in culture.
20. The non-embryonic stem, non-germ cells of 19 above that express one or more of oct4,
telomerase, rex-1, rox-1, or sox-2.
21. The non-embryonic stem, non-germ cells of 19 above that can differentiate into at least one
cell type of each of the endodermal, ectodermal, and mesodermal embryonic lineages.
22. The non-embryonic stem, non-germ cells of 21 above that express one or more of oct4,
telomerase, rex-1, rox-1, or sox-2.
In one embodiment, a conditioned medium is used instead of the stem cells.
In one embodiment, the organ is from a human.
In view of the property of the cells to achieve the desired effects, cell banks can be established
containing cells that are selected for having a desired potency (level of ability) to achieve the effects.
Accordingly, the invention encompasses assaying cells for the ability. The bank can provide a source for
making a pharmaceutical composition to administer to an organ. Cells can be used directly from the bank
or expanded prior to use. Especially in the case that the cells are subjected to further expansion, after
expansion it is desirable to validate that the cells still have the desired potency. Banks allow the “off the
shelf” use of cells that are allogeneic to the organ donor and recipient.
Accordingly, the invention also is directed to diagnostic procedures conducted prior to exposing
the stem cells to an organ. The procedures include assessing the potency of the cells to achieve the effects
described in this application. The cells may be taken from a cell bank and used directly or expanded prior
to administration. In either case, the cells could be assessed for the desired potency. Especially in the
case that the cells are subjected to further expansion, after expansion it is desirable to validate that the
cells still have the desired potency.
Although the cells selected for the effects are necessarily assayed during the selection procedure,
it may be preferable, and prudent, to again assay the cells prior to administration to a subject for treatment
to confirm that the cells still achieve the effects at desired levels. This is particularly preferable where the
cells have been stored for any length of time, such as in a cell bank, where cells are, most likely, frozen
during storage.
Between the original isolation of the cells and the administration to an organ, there may be
multiple (i.e., sequential) assays for the effects. This is to confirm that the cells can still achieve the
effects, at desired levels, after manipulations that occur within this time frame. For example, an assay
may be performed after each expansion of the cells. If cells are stored in a cell bank, they may be assayed
after being released from storage. If they are frozen, they may be assayed after thawing. If the cells from
a cell bank are expanded, they may be assayed after expansion. Preferably, a portion of the final cell
product (that is physically administered to the organ) may be assayed.
Since the stem cells may provide the effects described herein by means of secreted molecules,
the various embodiments described herein for administration of stem cells may be done by administration
of one or more of the secreted molecules, such as might be in conditioned culture medium.
The invention is also directed to compositions comprising a population of the cells having a
desired potency to achieve the desired effects. Such populations may be found as pharmaceutical
compositions suitable for administration to an organ and/or in cell banks from which cells can be used
directly for administration or expanded prior to administration. In one embodiment, the cells have
enhanced (increased) potency compared to the previous (parent) cell population. Parent cells are as
defined herein. Enhancement can be by selection of natural expressors or by external factors acting on
the cells.
The cells may be prepared by the isolation and culture conditions described herein. In a specific
embodiment, they are prepared by culture conditions that are described herein involving lower oxygen
concentrations combined with higher serum, such as those used to prepare the cells designated
“MultiStem®.”
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Schematic of study design.
Figure 2. Representative gross appearances of the right lower lobe (RLL) and left lower lobe
(LLL) of lung 2 following reperfusion. The MAPC-treated LLL appears normal while the vehicle-treated
RLL appears edematous and inflamed.
Figure 3. Semi-quantitative scoring demonstrates significant decrease in overall inflammation in
the MAPC-treated LLL compared to the vehicle-treated RLL in 4 out of 5 lungs and in aggregate. Means
+ SD of pooled observations from 3 blinded observers are depicted.
Figures 4A-B. Representative photomicrographs from lung 1 demonstrate minimal to no
significant inflammation in MAPC-treated LLL vs. alveolar septal thickening, edema, and perivascular
and peri-bronchial inflammatory cell infiltrates. Original Mag 200X.
Figures 5A-C. Decrease in total BAL fluid cell counts in the MAPC-treated LLL in lungs 3-5
(Fig. 5A). Total cell counts were not assessed in lungs 1 or 2. MAPC instillation also resulted in a
significant decreased in the elevated numbers of BAL fluid total neutrophils and eosinophils in all 5 lungs
(Figs. 5B-C). Data represents means + SEM of pooled observations from 3 blinded observers.
Figure 6. Representative BAL fluid cytokine analyses from Lung 4 demonstrate significant
increase in IL-10 in the MAPC-treated LLL but no significant change in iNOS, STC-1, or TSG-6. Data
represents means + standard deviations from triplicate determinations of each LLL or RLL sample.
Figure 7. Cytokine analysis of lung tissue. qPCR analysis was performed on the lung tissue
samples collected from the LLL and RLL of lungs 2-5at t=0, 2 and/or 4 hours. The fold-expression
represents the levels of the target gene compared to the t=0 value. All data were normalized to a
housekeeping gene, GAPDH. Data represents means + standard deviations from from LLL and RLL
samples from lungs 2-5.
DETAILED DESCRIPTION OF THE INVENTION
It should be understood that this invention is not limited to the particular methodology,
protocols, and reagents, etc., described herein and, as such, may vary. The terminology used herein is for
the purpose of describing particular embodiments only, and is not intended to limit the scope of the
disclosed invention, which is defined solely by the claims.
The section headings are used herein for organizational purposes only and are not to be
construed as in any way limiting the subject matter described.
The methods and techniques of the present application are generally performed according to
conventional methods well-known in the art and as described in various general and more specific
references that are cited and discussed throughout the present specification unless otherwise indicated.
See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (2001) and Ausubel et al., Current Protocols in Molecular
Biology, Greene Publishing Associates (1992), and Harlow and Lane, Antibodies: A Laboratory Manual,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990).
Definitions
“A” or “an” means herein one or more than one; at least one. Where the plural form is used
herein, it generally includes the singular.
A “cell bank” is industry nomenclature for cells that have been grown and stored for future use.
Cells may be stored in aliquots. They can be used directly out of storage or may be expanded after
storage. This is a convenience so that there are “off the shelf” cells available for administration. The
cells may already be stored in a pharmaceutically-acceptable excipient so they may be directly
administered or they may be mixed with an appropriate excipient when they are released from storage.
Cells may be frozen or otherwise stored in a form to preserve viability. In one embodiment of the
invention, cell banks are created in which the cells have been selected for enhanced potency to achieve
the effects described in this application. Following release from storage, and prior to administration, it
may be preferable to again assay the cells for potency. This can be done using any of the assays, direct or
indirect, described in this application or otherwise known in the art. Then cells having the desired
potency can then be administered. Banks can be made using autologous cells (derived from the organ
donor or recipient). Or banks can contain cells for allogeneic uses.
“Co-administer” means to administer in conjunction with one another, together, coordinately,
including simultaneous or sequential administration of two or more agents.
“Comprising” means, without other limitation, including the referent, necessarily, without any
qualification or exclusion on what else may be included. For example, “a composition comprising x and
y” encompasses any composition that contains x and y, no matter what other components may be present
in the composition. Likewise, “a method comprising the step of x” encompasses any method in which x
is carried out, whether x is the only step in the method or it is only one of the steps, no matter how many
other steps there may be and no matter how simple or complex x is in comparison to them. “Comprised
of and similar phrases using words of the root “comprise” are used herein as synonyms of “comprising”
and have the same meaning.
“Comprised of” is a synonym of “comprising” (see above).
The term “contact”, when used in relation to a stem cell and an organ to be transplanted, can
mean that, upon exposure to the organ, the stem cell physically touches the organ. In such instances, the
stem cell is in direct contact with the organ. In other instances, the stem cell can indirectly contact the
organ where one or more structures (e.g., another cell) and/or fluids (e.g., blood) physically intervene(s)
between the stem cell and the organ.
“EC cells” were discovered from analysis of a type of cancer called a teratocarcinoma. In 1964,
researchers noted that a single cell in teratocarcinomas could be isolated and remain undifferentiated in
culture. This type of stem cell became known as an embryonic carcinoma cell (EC cell).
“Effective amount” generally means an amount which provides the desired local or systemic
effect, e.g., effective to ameliorate undesirable effects of inflammation, including achieving the specific
desired effects described in this application. For example, an effective amount is an amount sufficient to
effectuate a beneficial or desired clinical result. The effective amounts can be provided all at once in a
single administration or in fractional amounts that provide the effective amount in several administrations.
The precise determination of what would be considered an effective amount may be based on factors
individual to each organ, including the type of organ, disease or injury being treated, the way the organ
has been processed, length of time from collection, etc. One skilled in the art will be able to determine
the effective amount for a given organ based on these considerations which are routine in the art. As used
herein, “effective dose” means the same as “effective amount.”
“Effective route” generally means a route which provides for delivery of an agent to a desired
compartment, system, or location. For example, an effective route is one through which an agent can be
administered to provide at the desired site of action an amount of the agent sufficient to effectuate a
beneficial or desired clinical result (in the present case, effective transplantation).
“Embryonic Stem Cells (ESC)” are well known in the art and have been prepared from many
different mammalian species. Embryonic stem cells are stem cells derived from the inner cell mass of an
early stage embryo known as a blastocyst. They are able to differentiate into all derivatives of the three
primary germ layers: ectoderm, endoderm, and mesoderm. These include each of the more than 220 cell
types in the adult body. The ES cells can become any tissue in the body, excluding placenta. Only the
morula’s cells are totipotent, able to become all tissues and a placenta. Some cells similar to ESCs may
be produced by nuclear transfer of a somatic cell nucleus into an enucleated fertilized egg.
The term “exogenous”, when used in relation to a stem cell, generally refers to a stem cell that is
external to the organ and which has been exposed to (e.g., contacted with) an organ intended for
transplantation by an effective route. An exogenous stem cell may be from the same subject or from a
different subject. In one embodiment, exogenous stem cells can include stem cells that have been
harvested from a subject, isolated, expanded ex vivo, and then exposed to an organ intended for
transplantation by an effective route.
The term “expose” can include the act of administering one or more stem cells to an organ
intended for transplantation. Administration to the organ can be done ex vivo or in vivo (e.g., by perfusion
into a subject).
Use of the term “includes” is not intended to be limiting.
“Increase” or “increasing” means to induce a biological event entirely or to increase the degree
of the event.
“Induced pluripotent stem cells (IPSC or IPS cells)” are somatic cells that have been
reprogrammed, for example, by introducing exogenous genes that confer on the somatic cell a less
differentiated phenotype. These cells can then be induced to differentiate into less differentiated progeny.
IPS cells have been derived using modifications of an approach originally discovered in 2006 (Yamanaka,
S. et al., Cell Stem Cell, 1:39-49 (2007)). For example, in one instance, to create IPS cells, scientists
started with skin cells that were then modified by a standard laboratory technique using retroviruses to
insert genes into the cellular DNA. In one instance, the inserted genes were Oct4, Sox2, Lif4, and c-myc,
known to act together as natural regulators to keep cells in an embryonic stem cell-like state. These cells
have been described in the literature. See, for example, Wernig et al., PNAS, 105:5856-5861 (2008);
Jaenisch et al., Cell, 132:567-582 (2008); Hanna et al., Cell, 133:250-264 (2008); and Brambrink et al.,
Cell Stem Cell, 2:151-159 (2008). These references are incorporated by reference for teaching IPSCs and
methods for producing them. It is also possible that such cells can be created by specific culture
conditions (exposure to specific agents).
The term “ischemic reperfusion injury” is understood in the industry and is described for
example in http://emedicine.medscape.com/article/431140-overview#aw2aab6b3 (about Organ
Preservation), as well as de Groot, H. et al., Transplant Proc. 39(2):481-4 (Mar. 2007), which are
incorporated herein by reference for the teaching of ischemic reperfusion injury and its mechanistic
details.
“Ischemia” occurs in two phases. The first phase is referred to as the warm ischemic phase and
includes the time from which the donor organ is removed and circulation is interrupted to the time that the
organ is administered with a hypothermic preservation solution. The cold ischemic phase occurs when
the organ is preserved in a hypothermic state prior to transplantation and normal recirculation in the
recipient.
The term “isolated” refers to a cell or cells which are not associated with one or more cells or
one or more cellular components that are associated with the cell or cells in vivo. An “enriched
population” means a relative increase in numbers of a desired cell relative to one or more other cell types
in vivo or in primary culture.
However, as used herein, the term “isolated” does not indicate the presence of only the cells of
the invention. Rather, the term “isolated” indicates that the cells of the invention are removed from their
natural tissue environment and are present at a higher concentration as compared to the normal tissue
environment. Accordingly, an “isolated” cell population may further include cell types in addition to the
cells of the invention cells and may include additional tissue components. This also can be expressed in
terms of cell doublings, for example. A cell may have undergone 10, 20, 30, 40 or more doublings in
vitro or ex vivo so that it is enriched compared to its original numbers in vivo or in its original tissue
environment (e.g., bone marrow, peripheral blood, placenta, umbilical cord, umbilical cord blood, adipose
tissue, etc.).
“MAPC” is an acronym for “multipotent adult progenitor cell.” It refers to a cell that is not an
embryonic stem cell or germ cell but has some characteristics of these. MAPC can be characterized in a
number of alternative descriptions, each of which conferred novelty to the cells when they were
discovered. They can, therefore, be characterized by one or more of those descriptions. First, they have
extended replicative capacity in culture without being transformed (tumorigenic) and with a normal
karyotype. Second, they may give rise to cell progeny of more than one germ layer, such as two or all
three germ layers (i.e., endoderm, mesoderm and ectoderm) upon differentiation. Third, although they are
not embryonic stem cells or germ cells, they may express markers of these primitive cell types so that
MAPCs may express one or more of Oct 3/4 (i.e., Oct 3A), rex-1, and rox-1. They may also express one
or more of sox-2 and SSEA-4. Fourth, like a stem cell, they may self-renew, that is, have an extended
replication capacity without being transformed. This means that these cells express telomerase (i.e., have
telomerase activity). Accordingly, the cell type that was designated “MAPC” may be characterized by
alternative basic characteristics that describe the cell via some of its novel properties.
The term “adult” in MAPC is non-restrictive. It refers to a non-embryonic somatic cell.
MAPCs are karyotypically normal and do not form teratomas in vivo. This acronym was first used in
U.S. Patent No. 7,015,037 to describe a pluripotent cell isolated from bone marrow. However, cells with
pluripotential markers and/or differentiation potential have been discovered subsequently and, for
purposes of this invention, may be equivalent to those cells first designated “MAPC.” Essential
descriptions of the MAPC type of cell are provided in the Summary of the Invention above.
MAPC represents a more primitive progenitor cell population than MSC (Verfaillie, C.M.,
Trends Cell Biol 12:502-8 (2002), Jahagirdar, B.N., et al., Exp Hematol, 29:543-56 (2001); Reyes, M. and
C.M. Verfaillie, Ann N Y Acad Sci, 938:231-233 (2001); Jiang, Y. et al., Exp Hematol, 30896-904 (2002);
and Jiang, Y. et al., Nature, 418:41-9. (2002)).
The term “MultiStem®” is the trade name for a cell preparation based on the MAPCs of U.S.
Patent No. 7,015,037, i.e., a non-embryonic stem, non-germ cell as described above. MultiStem® is
prepared according to cell culture methods disclosed in this patent application, particularly, lower oxygen
and higher serum. MultiStem® is highly expandable, karyotypically normal, and does not form teratomas
in vivo. It may differentiate into cell lineages of more than one germ layer and may express one or more
of telomerase, oct3/4, rex-1, rox-1, sox-2, and SSEA4.
The term “organ” may be used according to its customary and understood meaning in the
industry as an entire intact organ that has been removed from the donor for transplantation or is intended
to be removed from the donor for transplantation into a recipient. Although the term “organ” is
emphasized in this application, the methods apply to tissues that may not constitute whole organs. That
is, to parts of organs such as those disclosed elsewhere in this application. Therefore, where appropriate,
the term “tissue” can be appropriately substituted for the term “organ”.
“Pharmaceutically-acceptable carrier” is any pharmaceutically-acceptable medium for the cells
used in the present invention. Such a medium may retain isotonicity, cell metabolism, pH, and the like.
It is compatible with administration to an organ and can be used, therefore, for cell delivery and
treatment.
The term “potency” refers to the ability of the cells to achieve the effects described in this
application. Accordingly, potency refers to the effect at various levels, including, but not limited to,
increasing the probability of a successful transplantation, retarding the deterioration of a pre-transplant
organ, reducing inflammatory activity in the organ, providing immunological tolerance to the organ,
increasing the production of anti-inflammatory cytokines in the organ, increasing the presence of
neuroprotective T-cells in the organ, decreasing the presence of reactive T-cells in the organ, reducing the
level of pro-inflammatory cytokines in the organ, reducing the effects of hypoxia in the organ, reversing
the level of edema in the organ, and reducing the effects of hypothermia in the organ. Injury that is
sustained during recovery, preservation, and transplantation, occurs mainly from ischemia and
hypothermia. These can affect the organs in various ways. These are described in the Medscape
reference cited above and the link is given in this application. According to that reference, the
mechanisms of tissue injury include a loss of integrity in the cell structure, disruption of the ionic
composition of the cell, disruption in ATP generation, and, as a result of reperfusion, damage may occur
during reperfusion by the toxic accumulation of oxygen free radicals.
With respect to integrity of the cell structure, integrity may be interrupted by loss of structural
integrity in the cell membrane. Maintaining the integrity of the cell membrane depends on control of
temperature, pH, osmolarity. Organ ischemia and preservation disrupt all of these parameters.
“Primordial embryonic germ cells” (PG or EG cells) can be cultured and stimulated to produce
many less differentiated cell types.
“Progenitor cells” are cells produced during differentiation of a stem cell that have some, but
not all, of the characteristics of their terminally-differentiated progeny. Defined progenitor cells, such as
“cardiac progenitor cells,” are committed to a lineage, but not to a specific or terminally differentiated cell
type. The term “progenitor” as used in the acronym “MAPC” does not limit these cells to a particular
lineage. A progenitor cell can form a progeny cell that is more highly differentiated than the progenitor
cell.
The term “reduce” as used herein means to prevent as well as decrease. In the context of organ
treatment, to “reduce” is to either prevent or ameliorate organ rejection. This includes causes or
symptoms of organ rejection. This applies, for example, to the underlying biological cause of rejection,
such as, ameliorating the deleterious effects of inflammation.
“Selecting” a cell with a desired level of potency can mean identifying (as by assay), isolating,
and expanding a cell. This could create a population that has a higher potency than the parent cell
population from which the cell was isolated. The “parent” cell population refers to the parent cells from
which the selected cells divided. “Parent” refers to an actual P1 → F1 relationship (i.e., a progeny cell).
So if cell X is isolated from a mixed population of cells X and Y, in which X is an expressor and Y is not,
one would not classify a mere isolate of X as having enhanced expression. But, if a progeny cell of X is a
higher expressor, one would classify the progeny cell as having enhanced expression.
To select a cell that achieves the desired effect would include both an assay to determine if the
cells achieve the desired effect and would also include obtaining those cells. The cell may naturally
achieve the desired effect in that the effect is not achieved by an exogenous transgene/DNA. But an
effective cell may be improved by being incubated with or exposed to an agent that increases the effect.
The cell population from which the effective cell is selected may not be known to have the potency prior
to conducting the assay. The cell may not be known to achieve the desired effect prior to conducting the
assay. As an effect could depend on gene expression and/or secretion, one could also select on the basis
of one or more of the genes that cause the effect.
Selection could be from cells in a tissue. For example, in this case, cells would be isolated
from a desired tissue, expanded in culture, selected for achieving the desired effect, and the selected cells
further expanded.
Selection could also be from cells ex vivo, such as cells in culture. In this case, one or more of
the cells in culture would be assayed for achieving the desired effect and the cells obtained that achieve
the desired effect could be further expanded.
Cells could also be selected for enhanced ability to achieve the desired effect. In this case, the
cell population from which the enhanced cell is obtained already has the desired effect. Enhanced effect
means a higher average amount per cell than in the parent population.
The parent population from which the enhanced cell is selected may be substantially
homogeneous (the same cell type). One way to obtain such an enhanced cell from this population is to
create single cells or cell pools and assay those cells or cell pools to obtain clones that naturally have the
enhanced (greater) effect (as opposed to treating the cells with a modulator that induces or increases the
effect) and then expanding those cells that are naturally enhanced.
However, cells may be treated with one or more agents that will induce or increase the effect.
Thus, substantially homogeneous populations may be treated to enhance the effect.
If the population is not substantially homogeneous, then, it is preferable that the parental cell
population to be treated contains at least 100 of the desired cell type in which enhanced effect is sought,
more preferably at least 1,000 of the cells, and still more preferably, at least 10,000 of the cells.
Following treatment, this sub-population can be recovered from the heterogeneous population by known
cell selection techniques and further expanded if desired.
Thus, desired levels of effect may be those that are higher than the levels in a given preceding
population. For example, cells that are put into primary culture from a tissue and expanded and isolated
by culture conditions that are not specifically designed to produce the effect may provide a parent
population. Such a parent population can be treated to enhance the average effect per cell or screened for
a cell or cells within the population that express greater degrees of effect without deliberate treatment.
Such cells can be expanded then to provide a population with a higher (desired) expression.
“Self-renewal” of a stem cell refers to the ability to produce replicate daughter stem cells
having differentiation potential that is identical to those from which they arose. A similar term used in
this context is “proliferation.”
“Stem cell” means a cell that can undergo self-renewal (i.e., progeny with the same
differentiation potential) and also produce progeny cells that are more restricted in differentiation
potential. Within the context of the invention, a stem cell would also encompass a more differentiated
cell that has de-differentiated, for example, by nuclear transfer, by fusion with a more primitive stem cell,
by introduction of specific transcription factors, or by culture under specific conditions. See, for example,
Wilmut et al., Nature, 385:810-813 (1997); Ying et al., Nature, 416:545-548 (2002); Guan et al., Nature,
440:1199-1203 (2006); Takahashi et al., Cell, 126:663-676 (2006); Okita et al., Nature, 448:313-317
(2007); and Takahashi et al., Cell, 131:861-872 (2007).
Dedifferentiation may also be caused by the administration of certain compounds or exposure
to a physical environment in vitro or in vivo that would cause the dedifferentiation. Stem cells also may
be derived from abnormal tissue, such as a teratocarcinoma and some other sources such as embryoid
bodies (although these can be considered embryonic stem cells in that they are derived from embryonic
tissue, although not directly from the inner cell mass). Stem cells may also be produced by introducing
genes associated with stem cell function into a non-stem cell, such as an induced pluripotent stem cell.
“Subject” means a vertebrate, such as a mammal, such as a human. Mammals include, but are
not limited to, humans, dogs, cats, horses, cows, and pigs.
The term “therapeutically effective amount” refers to the amount of an agent determined to
produce any therapeutic response in a mammal. For example, effective anti-inflammatory therapeutic
agents may prolong the survivability of the patient, and/or inhibit overt clinical symptoms. Treatments
that are therapeutically effective within the meaning of the term as used herein, include treatments that
improve a subject’s quality of life even if they do not improve the disease outcome per se. Such
therapeutically effective amounts are readily ascertained by one of ordinary skill in the art. Thus, to
“treat” means to deliver such an amount. Thus, treating can prevent or ameliorate any pathological
symptoms.
The term “tolerization” or “tolerize” refers to the treatment of the pre-transplantation organ
(graft) with the stem cells to reduce the immunogenicity of the graft to enable or facilitate the
development of tolerance of the organ by the recipient. The term broadly refers to the concept of
reducing the immunogenicity of the transplant organ, which enables or facilitates tolerance development
by the recipient. Thus, tolerizing the organ causes the organ to be tolerated by the recipient. In other
words, the term can refer to making the immune system unable to elicit an immune response to a cell or
tissue that normally elicits an immune response. An example of this is when a T-regulatory cell secretes
factors that suppress an activated T-cell so that I can no longer secreted pro-inflammatory cytokines.
This might be accomplished via ex vivo treatment prior to transplantation, or even local
administration prior to harvest.
“Treat,” “treating,” or “treatment” are used broadly in relation to the invention and each such
term encompasses, among others, preventing, ameliorating, inhibiting, or curing a deficiency,
dysfunction, disease, or other deleterious process, including those that interfere with and/or result from a
therapy.
“Validate” means to confirm. In the context of the invention, one confirms that a cell is an
expressor with a desired potency. This is so that one can then use that cell (in treatment, banking, drug
screening, etc.) with a reasonable expectation of efficacy. Accordingly, to validate means to confirm that
the cells, having been originally found to have/established as having the desired activity, in fact, retain
that activity. Thus, validation is a verification event in a two-event process involving the original
determination and the follow-up determination. The second event is referred to herein as “validation.”
Stem Cells
The present invention can be practiced, preferably, using stem cells of vertebrate species, such
as humans, non-human primates, domestic animals, livestock, and other non-human mammals. These
include, but are not limited to, those cells described below.
Embryonic Stem Cells
The most well studied stem cell is the embryonic stem cell (ESC) as it has unlimited self-
renewal and multipotent differentiation potential. These cells are derived from the inner cell mass of the
blastocyst or can be derived from the primordial germ cells of a post-implantation embryo (embryonal
germ cells or EG cells). ES and EG cells have been derived, first from mouse, and later, from many
different animals, and more recently, also from non-human primates and humans. When introduced into
mouse blastocysts or blastocysts of other animals, ESCs can contribute to all tissues of the animal. ES
and EG cells can be identified by positive staining with antibodies against SSEA1 (mouse) and SSEA4
(human). See, for example, U.S. Patent Nos. 5,453,357; 5,656,479; 5,670,372; 5,843,780; 5,874,301;
,914,268; 6,110,739 6,190,910; 6,200,806; 6,432,711; 6,436,701, 6,500,668; 6,703,279; 6,875,607;
7,029,913; 7,112,437; 7,145,057; 7,153,684; and 7,294,508, each of which is incorporated by reference
for teaching embryonic stem cells and methods of making and expanding them. Accordingly, ESCs and
methods for isolating and expanding them are well-known in the art.
A number of transcription factors and exogenous cytokines have been identified that influence
the potency status of embryonic stem cells in vivo. The first transcription factor to be described that is
involved in stem cell pluripotency is Oct4. Oct4 belongs to the POU (Pit-Oct-Unc) family of
transcription factors and is a DNA binding protein that is able to activate the transcription of genes,
containing an octameric sequence called “the octamer motif” within the promoter or enhancer region.
Oct4 is expressed at the moment of the cleavage stage of the fertilized zygote until the egg cylinder is
formed. The function of Oct3/4 is to repress differentiation inducing genes (i.e., FoxaD3, hCG) and to
activate genes promoting pluripotency (FGF4, Utf1, Rex1). Sox2, a member of the high mobility group
(HMG) box transcription factors, cooperates with Oct4 to activate transcription of genes expressed in the
inner cell mass. It is essential that Oct3/4 expression in embryonic stem cells is maintained between
certain levels. Overexpression or downregulation of >50% of Oct4 expression level will alter embryonic
stem cell fate, with the formation of primitive endoderm/mesoderm or trophectoderm, respectively. In
vivo, Oct4 deficient embryos develop to the blastocyst stage, but the inner cell mass cells are not
pluripotent. Instead they differentiate along the extraembryonic trophoblast lineage. Sall4, a mammalian
Spalt transcription factor, is an upstream regulator of Oct4, and is therefore important to maintain
appropriate levels of Oct4 during early phases of embryology. When Sall4 levels fall below a certain
threshold, trophectodermal cells will expand ectopically into the inner cell mass. Another transcription
factor required for pluripotency is Nanog, named after a celtic tribe “Tir Nan Og”: the land of the ever
young. In vivo, Nanog is expressed from the stage of the compacted morula, is subsequently defined to
the inner cell mass and is downregulated by the implantation stage. Downregulation of Nanog may be
important to avoid an uncontrolled expansion of pluripotent cells and to allow multilineage differentiation
during gastrulation. Nanog null embryos, isolated at day 5.5, consist of a disorganized blastocyst, mainly
containing extraembryonic endoderm and no discernible epiblast.
Non-Embryonic Stem Cells
Stem cells have been identified in most tissues. Perhaps the best characterized is the
hematopoietic stem cell (HSC). HSCs are mesoderm-derived cells that can be purified using cell surface
markers and functional characteristics. They have been isolated from bone marrow, peripheral blood,
cord blood, fetal liver, and yolk sac. They initiate hematopoiesis and generate multiple hematopoietic
lineages. When transplanted into lethally-irradiated animals, they can repopulate the erythroid neutrophil-
macrophage, megakaryocyte, and lymphoid hematopoietic cell pool. They can also be induced to
undergo some self-renewal cell division. See, for example, U.S. Patent Nos. 5,635,387; 5,460,964;
,677,136; 5,750,397; 5,681,599; and 5,716,827. U.S. Patent No. 5,192,553 reports methods for isolating
human neonatal or fetal hematopoietic stem or progenitor cells. U.S. Patent No. 5,716,827 reports human
hematopoietic cells that are Thy-1+ progenitors, and appropriate growth media to regenerate them in
vitro. U.S. Patent No. 5,635,387 reports a method and device for culturing human hematopoietic cells
and their precursors. U.S. Patent No. 6,015,554 describes a method of reconstituting human lymphoid
and dendritic cells. Accordingly, HSCs and methods for isolating and expanding them are well-known in
the art.
Another stem cell that is well-known in the art is the neural stem cell (NSC). These cells can
proliferate in vivo and continuously regenerate at least some neuronal cells. When cultured ex vivo,
neural stem cells can be induced to proliferate as well as differentiate into different types of neurons and
glial cells. When transplanted into the brain, neural stem cells can engraft and generate neural and glial
cells. See, for example, Gage F.H., Science, 287:1433-1438 (2000), Svendsen S.N. et al., Brain
Pathology, 9:499-513 (1999), and Okabe S. et al., Mech Development, 59:89-102 (1996). U.S. Patent No.
,851,832 reports multipotent neural stem cells obtained from brain tissue. U.S. Patent No. 5,766,948
reports producing neuroblasts from newborn cerebral hemispheres. U.S. Patent Nos. 5,564,183 and
,849,553 report the use of mammalian neural crest stem cells. U.S. Patent No. 6,040,180 reports in vitro
generation of differentiated neurons from cultures of mammalian multipotential CNS stem cells. WO
98/50526 and WO 99/01159 report generation and isolation of neuroepithelial stem cells,
oligodendrocyte-astrocyte precursors, and lineage-restricted neuronal precursors. U.S. Patent No.
,968,829 reports neural stem cells obtained from embryonic forebrain. Accordingly, neural stem cells
and methods for making and expanding them are well-known in the art.
Another stem cell that has been studied extensively in the art is the mesenchymal stem cell
(MSC). MSCs are derived from the embryonal mesoderm and can be isolated from many sources,
including adult bone marrow, peripheral blood, fat, placenta, and umbilical blood, among others. MSCs
can differentiate into many mesodermal tissues, including muscle, bone, cartilage, fat, and tendon. There
is considerable literature on these cells. See, for example, U.S. Patent Nos. 5,486,389; 5,827,735;
,811,094; 5,736,396; 5,837,539; 5,837,670; and 5,827,740. See also Pittenger, M. et al., Science,
284:143-147 (1999).
Another example of an adult stem cell is adipose-derived adult stem cells (ADSCs) which have
been isolated from fat, typically by liposuction followed by release of the ADSCs using collagenase.
ADSCs are similar in many ways to MSCs derived from bone marrow, except that it is possible to isolate
many more cells from fat. These cells have been reported to differentiate into bone, fat, muscle, cartilage,
and neurons. A method of isolation has been described in U.S. Patent Publication No. 2005/0153442 A1.
Other stem cells that are known in the art include gastrointestinal stem cells, epidermal stem
cells, and hepatic stem cells, which have also been termed “oval cells” (Potten, C., et al., Trans R Soc
Lond B Biol Sci, 353:821-830 (1998), Watt, F., Trans R Soc Lond B Biol Sci, 353:831 (1997); Alison et
al., Hepatology, 29:678-683 (1998).
Other non-embryonic cells reported to be capable of differentiating into cell types of more than
one embryonic germ layer include, but are not limited to, cells from umbilical cord blood (see U.S.
Publication No. 2002/0164794), placenta (see U.S. Publication No. 2003/0181269, umbilical cord matrix
(Mitchell, K.E. et al., Stem Cells, 21:50-60 (2003)), small embryonic-like stem cells (Kucia, M. et al., J
Physiol Pharmacol, 57 Suppl 5:5-18 (2006)), amniotic fluid stem cells (Atala, A., J Tissue Regen Med,
1:83-96 (2007)), skin-derived precursors (Toma et al., Nat Cell Biol, 3:778-784 (2001)), bone marrow
(see U.S. Patent Publication Nos. 2003/0059414 and 2006/0147246), marrow-isolated adult multilineage
inducible (MIAMI) cells (see ), and endometrial cells (see U.S. Publication No.
2013/0156726), each of which is incorporated by reference for teaching these cells.
Strategies of Reprogramming Somatic Cells
Several different strategies, such as nuclear transplantation, cellular fusion, and culture induced
reprogramming have been employed to induce the conversion of differentiated cells into an embryonic
state. Nuclear transfer involves the injection of a somatic nucleus into an enucleated oocyte, which, upon
transfer into a surrogate mother, can give rise to a clone (“reproductive cloning”), or, upon explantation in
culture, can give rise to genetically matched embryonic stem (ES) cells (“somatic cell nuclear transfer,”
SCNT). Cell fusion of somatic cells with ES cells results in the generation of hybrids that show all
features of pluripotent ES cells. Explantation of somatic cells in culture selects for immortal cell lines
that may be pluripotent or multipotent. At present, spermatogonial stem cells are the only source of
pluripotent cells that can be derived from postnatal animals. Transduction of somatic cells with defined
factors can initiate reprogramming to a pluripotent state. These experimental approaches have been
extensively reviewed (Hochedlinger and Jaenisch, Nature, 441:1061-1067 (2006) and Yamanaka, S., Cell
Stem Cell, 1:39-49 (2007)).
Nuclear Transfer
Nuclear transplantation (NT), also referred to as somatic cell nuclear transfer (SCNT), denotes
the introduction of a nucleus from a donor somatic cell into an enucleated ogocyte to generate a cloned
animal such as Dolly the sheep (Wilmut et al., Nature, 385:810-813 (1997). The generation of live
animals by NT demonstrated that the epigenetic state of somatic cells, including that of terminally
differentiated cells, while stable, is not irreversible fixed but can be reprogrammed to an embryonic state
that is capable of directing development of a new organism. In addition to providing an exciting
experimental approach for elucidating the basic epigenetic mechanisms involved in embryonic
development and disease, nuclear cloning technology is of potential interest for patient-specific
transplantation medicine.
Fusion of Somatic Cells and Embryonic Stem Cells
Epigenetic reprogramming of somatic nuclei to an undifferentiated state has been demonstrated
in murine hybrids produced by fusion of embryonic cells with somatic cells. Hybrids between various
somatic cells and embryonic carcinoma cells (Solter, D., Nat Rev Genet, 7:319-327 (2006), embryonic
germ (EG), or ES cells (Zwaka and Thomson, Development, 132:227-233 (2005)) share many features
with the parental embryonic cells, indicating that the pluripotent phenotype is dominant in such fusion
products. As with mouse (Tada et al., Curr Biol, 11:1553-1558 (2001)), human ES cells have the
potential to reprogram somatic nuclei after fusion (Cowan et al., Science, 309:1369-1373(2005)); Yu et
al., Science, 318:1917-1920 (2006)). Activation of silent pluripotency markers such as Oct4 or
reactivation of the inactive somatic X chromosome provided molecular evidence for reprogramming of
the somatic genome in the hybrid cells. It has been suggested that DNA replication is essential for the
activation of pluripotency markers, which is first observed 2 days after fusion (Do and Scholer, Stem
Cells, 22:941-949 (2004)), and that forced overexpression of Nanog in ES cells promotes pluripotency
when fused with neural stem cells (Silva et al., Nature, 441:997-1001 (2006)).
Culture-Induced Reprogramming
Pluripotent cells have been derived from embryonic sources such as blastomeres and the inner
cell mass (ICM) of the blastocyst (ES cells), the epiblast (EpiSC cells), primordial germ cells (EG cells),
and postnatal spermatogonial stem cells (“maGSCsm” “ES-like” cells). The following pluripotent cells,
along with their donor cell/tissue is as follows: parthogenetic ES cells are derived from murine oocytes
(Narasimha et al., Curr Biol, 7:881-884 (1997)); embryonic stem cells have been derived from
blastomeres (Wakayama et al., Stem Cells, 25:986-993 (2007)); inner cell mass cells (source not
applicable) (Eggan et al., Nature, 428:44-49 (2004)); embryonic germ and embryonal carcinoma cells
have been derived from primordial germ cells (Matsui et al., Cell, 70:841-847 (1992)); GMCS, maSSC,
and MASC have been derived from spermatogonial stem cells (Guan et al., Nature, 440:1199-1203
(2006); Kanatsu-Shinohara et al., Cell, 119:1001-1012 (2004); and Seandel et al., Nature, 449:346-350
(2007)); EpiSC cells are derived from epiblasts (Brons et al., Nature, 448:191-195 (2007); Tesar et al.,
Nature, 448:196-199(2007)); parthogenetic ES cells have been derived from human oocytes (Cibelli et
al., Science, 295L819 (2002); Revazova et al., Cloning Stem Cells, 9:432-449 (2007)); human ES cells
have been derived from human blastocysts (Thomson et al., Science, 282:1145-1147 (1998)); MAPC
have been derived from bone marrow (Jiang et al., Nature, 418:41-49 (2002); Phinney and Prockop, Stem
Cells, 25:2896-2902 (2007)); cord blood cells (derived from cord blood) (van de Ven et al., Exp Hematol,
:1753-1765 (2007)); neurosphere derived cells derived from neural cell (Clarke et al., Science,
288:1660-1663 (2000)). Donor cells from the germ cell lineage such as PGCs or spermatogonial stem
cells are known to be unipotent in vivo, but it has been shown that pluripotent ES-like cells (Kanatsu-
Shinohara et al., Cell, 119:1001-1012 (2004) or maGSCs (Guan et al., Nature, 440:1199-1203 (2006),
can be isolated after prolonged in vitro culture. While most of these pluripotent cell types were capable
of in vitro differentiation and teratoma formation, only ES, EG, EC, and the spermatogonial stem cell-
derived maGCSs or ES-like cells were pluripotent by more stringent criteria, as they were able to form
postnatal chimeras and contribute to the germline. Recently, multipotent adult spermatogonial stem cells
(MASCs) were derived from testicular spermatogonial stem cells of adult mice, and these cells had an
expression profile different from that of ES cells (Seandel et al., Nature, 449:346-350 (2007)) but similar
to EpiSC cells, which were derived from the epiblast of postimplantation mouse embryos (Brons et al.,
Nature, 448:191-195 (2007); Tesar et al., Nature, 448:196-199 (2007)).
Reprogramming by Defined Transcription Factors
Takahashi and Yamanaka have reported reprogramming somatic cells back to an ES-like state
(Takahashi and Yamanaka, Cell, 126:663-676 (2006)). They successfully reprogrammed mouse
embryonic fibroblasts (MEFs) and adult fibroblasts to pluripotent ES-like cells after viral-mediated
transduction of the four transcription factors Oct4, Sox2, c-myc, and Klf4 followed by selection for
activation of the Oct4 target gene Fbx15. Cells that had activated Fbx15 were coined iPS (induced
pluripotent stem) cells and were shown to be pluripotent by their ability to form teratomas, although they
were unable to generate live chimeras. This pluripotent state was dependent on the continuous viral
expression of the transduced Oct4 and Sox2 genes, whereas the endogenous Oct4 and Nanog genes were
either not expressed or were expressed at a lower level than in ES cells, and their respective promoters
were found to be largely methylated. This is consistent with the conclusion that the Fbx15-iPS cells did
not correspond to ES cells but may have represented an incomplete state of reprogramming. While
genetic experiments had established that Oct4 and Sox2 are essential for pluripotency (Chambers and
Smith, Oncogene, 23:7150-7160 (2004); Ivanona et al., Nature, 442:5330538 (2006); Masui et al., Nat
Cell Biol, 9:625-635 (2007)), the role of the two oncogenes c-myc and Klf4 in reprogramming is less
clear. Some of these oncogenes may, in fact, be dispensable for reprogramming, as both mouse and
human iPS cells have been obtained in the absence of c-myc transduction, although with low efficacy
(Nakagawa et al., Nat Biotechnol, 26:191-106 (2008); Werning et al., Nature, 448:318-324 (2008); Yu et
al., Science, 318: 1917-1920 (2007)).
MAPC
Human MAPCs are described in U.S. Patent 7,015,037. MAPCs have been identified in other
mammals. Murine MAPCs, for example, are also described in U.S. Patent 7,015,037. Rat MAPCs are
also described in U.S. Patent No. 7,838,289.
These references are incorporated by reference for describing MAPCs first isolated by
Catherine Verfaillie.
Isolation and Growth of MAPCs
Methods of MAPC isolation are known in the art. See, for example, U.S. Patent 7,015,037, and
these methods, along with the characterization (phenotype) of MAPCs, are incorporated herein by
reference. MAPCs can be isolated from multiple sources, including, but not limited to, bone marrow,
placenta, umbilical cord and cord blood, muscle, brain, liver, spinal cord, blood or skin. It is, therefore,
possible to obtain bone marrow aspirates, brain or liver biopsies, and other organs, and isolate the cells
using positive or negative selection techniques available to those of skill in the art, relying upon the genes
that are expressed (or not expressed) in these cells (e.g., by functional or morphological assays such as
those disclosed in the above-referenced applications, which have been incorporated herein by reference).
MAPCs have also been obtained my modified methods described in Breyer et al., Experimental
Hematology, 34:1596-1601 (2006) and Subramanian et al., Cellular Programming and Reprogramming:
Methods and Protocols; S. Ding (ed.), Methods in Molecular Biology, 636:55-78 (2010), incorporated by
reference for these methods.
MAPCs from Human Bone Marrow as Described in U.S. Patent 7,015,037
MAPCs do not express the common leukocyte antigen CD45 or erythroblast specific
glycophorin-A (Gly-A). The mixed population of cells was subjected to a Ficoll Hypaque separation. The
cells were then subjected to negative selection using anti-CD45 and anti-Gly-A antibodies, depleting the
population of CD45+ and Gly-A+ cells, and the remaining approximately 0.1% of marrow mononuclear
cells were then recovered. Cells could also be plated in fibronectin-coated wells and cultured as
described below for 2-4 weeks to deplete the cells of CD45+ and Gly-A+ cells. In cultures of adherent
bone marrow cells, many adherent stromal cells undergo replicative senescence around cell doubling 30
and a more homogenous population of cells continues to expand and maintains long telomeres.
Alternatively, positive selection could be used to isolate cells via a combination of cell-specific
markers. Both positive and negative selection techniques are available to those of skill in the art, and
numerous monoclonal and polyclonal antibodies suitable for negative selection purposes are also
available in the art (see, for example, Leukocyte Typing V, Schlossman, et al., Eds. (1995) Oxford
University Press) and are commercially available from a number of sources.
Techniques for mammalian cell separation from a mixture of cell populations have also been
described by Schwartz, et al., in U. S. Patent No. 5,759,793 (magnetic separation), Basch et al., 1983
(immunoaffinity chromatography), and Wysocki and Sato, 1978 (fluorescence-activated cell sorting).
Cells may be cultured in low-serum or serum-free culture medium. Serum-free medium used
to culture MAPCs is described in U.S. Patent 7,015,037. Commonly-used growth factors include but are
not limited to platelet-derived growth factor and epidermal growth factor. See, for example, U.S. Patent
Nos. 7,169,610; 7,109,032; 7,037,721; 6,617,161; 6,617,159; 6,372,210;6,224,860; 6,037,174; 5,908,782;
,766,951; 5,397,706; and 4,657,866; all incorporated by reference for teaching growing cells in serum-
free medium.
Additional Culture Methods
In additional experiments, the density at which MAPCs are cultured can vary from about 100
2 2 2 2
cells/cm or about 150 cells/cm to about 10,000 cells/cm , including about 200 cells/cm to about 1500
cells/cm to about 2000 cells/cm . The density can vary between species. Additionally, optimal density
can vary depending on culture conditions and source of cells. It is within the skill of the ordinary artisan
to determine the optimal density for a given set of culture conditions and cells.
Also, effective atmospheric oxygen concentrations of less than about 10%, including about 1-
% and, especially, 3-5%, can be used at any time during the isolation, growth and differentiation of
MAPCs in culture.
Cells may be cultured under various serum concentrations, e.g., about 2-20%. Fetal bovine
serum may be used. Higher serum may be used in combination with lower oxygen tensions, for example,
about 15-20%. Cells need not be selected prior to adherence to culture dishes. For example, after a Ficoll
gradient, cells can be directly plated, e.g., 250,000-500,000/cm . Adherent colonies can be picked,
possibly pooled, and expanded.
In one embodiment, used in the experimental procedures in the Examples, high serum (around
-20%) and low oxygen (around 3-5%) conditions were used for the cell culture. Specifically, adherent
cells from colonies were plated and passaged at densities of about 1700-2300 cells/cm in 18% serum and
3% oxygen (with PDGF and EGF).
In an embodiment specific for MAPCs, supplements are cellular factors or components that
allow MAPCs to retain the ability to differentiate into cell types of more than one embryonic lineage,
such as all three lineages. This may be indicated by the expression of specific markers of the
undifferentiated state, such as Oct 3/4 (Oct 3A) and/or markers of high expansion capacity, such as
telomerase.
Cell Culture
For all the components listed below, see U.S. 7,015,037, which is incorporated by reference for
teaching these components.
In general, cells useful for the invention can be maintained and expanded in culture medium
that is available and well-known in the art. Also contemplated is supplementation of cell culture medium
with mammalian sera. Additional supplements can also be used advantageously to supply the cells with
the necessary trace elements for optimal growth and expansion. Hormones can also be advantageously
used in cell culture. Lipids and lipid carriers can also be used to supplement cell culture media,
depending on the type of cell and the fate of the differentiated cell. Also contemplated is the use of feeder
cell layers.
Cells in culture can be maintained either in suspension or attached to a solid support, such as
extracellular matrix components. Stem cells often require additional factors that encourage their
attachment to a solid support, such as type I and type II collagen, chondroitin sulfate, fibronectin,
“superfibronectin” and fibronectin-like polymers, gelatin, poly-D and poly-L-lysine, thrombospondin and
vitronectin. One embodiment of the present invention utilizes fibronectin. See, for example, Ohashi et
al., Nature Medicine, 13:880-885 (2007); Matsumoto et al., J Bioscience and Bioengineering, 105:350-
354 (2008); Kirouac et al., Cell Stem Cell, 3:369-381 (2008); Chua et al., Biomaterials, 26:2537-2547
(2005); Drobinskaya et al., Stem Cells, 26:2245-2256 (2008); Dvir-Ginzberg et al., FASEB J, 22:1440-
1449 (2008); Turner et al., J Biomed Mater Res Part B: Appl Biomater, 82B:156-168 (2007); and
Miyazawa et al., Journal of Gastroenterology and Hepatology, 22:1959-1964 (2007)).
Cells may also be grown in “3D” (aggregated) cultures. An example is ,
filed January 21, 2009.
Once established in culture, cells can be used fresh or frozen and stored as frozen stocks, using,
for example, DMEM with 40% FCS and 10% DMSO. Other methods for preparing frozen stocks for
cultured cells are also available to those of skill in the art.
Pharmaceutical Formulations
U.S. 7,015,037 is incorporated by reference for teaching pharmaceutical formulations. In
certain embodiments, the cell populations are present within a composition adapted for and suitable for
delivery, i.e., physiologically compatible.
Formulations would be oriented to degree of desired effect, such as reduction of inflammation,
reduction of apoptosis, edema, etc., upregulation of certain factors, etc.
In some embodiments the purity of the cells for administration to an organ is about 100%
(substantially homogeneous). In other embodiments it is 95% to 100%. In some embodiments it is 85%
to 95%. Particularly, in the case of admixtures with other cells, the percentage can be about 10%-15%,
%-20%, 20%-25%, 25%-30%, 30%-35%, 35%-40%, 40%-45%, 45%-50%, 60%-70%, 70%-80%,
80%-90%, or 90%-95%. Or isolation/purity can be expressed in terms of cell doublings where the cells
have undergone, for example, 10-20, 20-30, 30-40, 40-50 or more cell doublings.
The choice of formulation for administering the cells will depend on a variety of factors.
Prominent among these will be the species of donor/recipient, the nature of the organ being treated, the
nature of other therapies and agents that are being administered, the optimum route for administration,
survivability via the effective route, the dosing regimen, and other factors that will be apparent to those
skilled in the art. For instance, the choice of suitable carriers and other additives will depend on the exact
route of administration and the nature of the particular dosage form.
Final formulations of the aqueous suspension of cells/medium will typically involve adjusting
the ionic strength of the suspension to isotonicity (i.e., about 0.1 to 0.2) and to physiological pH (i.e.,
about pH 6.8 to 7.5). The final formulation will also typically contain a fluid lubricant.
The skilled artisan can readily determine the amount of cells and optional additives, vehicles,
and/or carrier in compositions to be administered in methods of the invention. Typically, any additives
(in addition to the cells) are present in an amount of 0.001 to 50 wt % in solution, such as in phosphate
buffered saline. The active ingredient is present in the order of micrograms to milligrams, such as about
0.0001 to about 5 wt %, preferably about 0.0001 to about 1 wt %, most preferably about 0.0001 to about
0.05 wt % or about 0.001 to about 20 wt %, preferably about 0.01 to about 10 wt %, and most preferably
about 0.05 to about 5 wt %.
In some embodiments cells are encapsulated for administration, particularly where
encapsulation enhances the effectiveness or provides advantages in handling and/or shelf life. Cells may
be encapsulated by membranes, as well as capsules. It is contemplated that any of the many methods of
cell encapsulation available may be employed.
A wide variety of materials may be used in various embodiments for microencapsulation of
cells. Such materials include, for example, polymer capsules, alginate-poly-L-lysine-alginate
microcapsules, barium poly-L-lysine alginate capsules, barium alginate capsules,
polyacrylonitrile/polyvinylchloride (PAN/PVC) hollow fibers, and polyethersulfone (PES) hollow fibers.
Techniques for microencapsulation of cells that may be used for administration of cells are
known to those of skill in the art and are described, for example, in Chang, P., et al., 1999; Matthew,
H.W., et al., 1991; Yanagi, K., et al., 1989; Cai Z.H., et al., 1988; Chang, T.M., 1992 and in U.S. Patent
No. 5,639,275 (which, for example, describes a biocompatible capsule for long-term maintenance of cells
that stably express biologically active molecules). Additional methods of encapsulation are in European
Patent Publication No. 301,777 and U.S. Pat. Nos. 4,353,888; 4,744,933; 4,749,620; 4,814,274;
,084,350; 5,089,272; 5,578,442; 5,639,275; and 5,676,943. All of the foregoing are incorporated herein
by reference in parts pertinent to encapsulation of cells.
Certain embodiments incorporate cells into a polymer, such as a biopolymer or synthetic
polymer. Examples of biopolymers include, but are not limited to, fibronectin, fibrin, fibrinogen,
thrombin, collagen, and proteoglycans. Other factors, such as the cytokines discussed above, can also be
incorporated into the polymer. In other embodiments of the invention, cells may be incorporated in the
interstices of a three-dimensional gel. A large polymer or gel, typically, will be surgically implanted. A
polymer or gel that can be formulated in small enough particles or fibers can be administered by other
common, more convenient, non-surgical routes.
The dosage of the cells will vary within wide limits and will be fitted to the individual
requirements in each particular case. The number of cells will vary depending on the type, weight, and
condition of the organ, the number or frequency of administrations, and other variables known to those of
skill in the art. The cells can be administered by a route that is suitable for the tissue or organ. Examples
of suitable delivery routes can include intra-tracheal delivery (e.g., for lung), intravenous delivery, intra-
arterial delivery (e.g., intra-coronary), direct injection into the organ, and intra-lymphatic system delivery.
The cells can be suspended in an appropriate excipient in a concentration from about 0.01 to 1
5 6 6 7
x 10 cells/ml, about 1 x 10 cells/ml to 10 x 10 cells/ml, or about 10 x 10 cells/ml to 5 x10 cells/ml.
Suitable excipients are those that are biologically and physiologically compatible with the cells and with
the recipient organ, such as buffered saline solution or other suitable excipients. The composition for
administration can be formulated, produced, and stored according to standard methods complying with
proper sterility and stability.
Dosing
Doses (i.e., the number of cells) for humans or other mammals can be determined without
undue experimentation by the skilled artisan, from this disclosure, the documents cited herein, and the
knowledge in the art. The optimal dose to be used in accordance with various embodiments of the
invention will depend on numerous factors, including the following: the disease being treated and its
stage; the species of the donor, their health, gender, age, weight, and metabolic rate; the donor’s
immunocompetence; other therapies being administered; and expected potential complications from the
donor’s history or genotype. The parameters may also include: whether the cells are syngeneic,
autologous, allogeneic, or xenogeneic; their potency; the site and/or distribution that must be targeted; and
such characteristics of the site such as accessibility to cells. Additional parameters include co-
administration with other factors (such as growth factors and cytokines). The optimal dose in a given
situation also will take into consideration the way in which the cells are formulated, the way they are
administered (e.g., perfusion, intra-organ, etc.), and the degree to which the cells will be localized at the
target sites following administration.
Ultimately, the dose levels, timing, and frequency would be determined by effectiveness. This
will be measured by organ health and viability, and possibly by organ function and clinical measures
post-transplant. Such measures will vary by organ. In one embodiment, they could include organ
function measures. One could access measures of organ viability for transplant via the clinical literature.
In another embodiment, the level(s) or pattern(s) of certain markers (e.g., tissue mRNA levels, cytokine
levels, and inflammatory cell numbers) can be assayed (e.g., using qPCR) to determine effectiveness. For
example, the level of IL-10 mRNA from pulmonary tissue can be assayed by qPCR to determine
effectiveness. In another example, one might evaluate dose in lung by impact on: cytokine levels; other
inflammatory markers in fluids (e.g., obtained by bronchoalveolar lavage); edema levels; hemodynamic
and ventilator measures; and evaluation of gas exchange in ex vivo (re)perfused lungs.
In various embodiments, cells/medium may be administered in an initial dose, and thereafter
maintained by further administration. Cells may be administered by one method initially, and thereafter
administered by the same method or one or more different methods. The levels can be maintained by the
ongoing administration of the cells/medium. Various embodiments administer the cells either initially or
to maintain their level in the subject or both by intravenous injection. In a variety of embodiments, other
forms of administration are used, dependent upon the type and condition of the organ and other factors,
discussed elsewhere herein.
Cells/medium may be administered in many frequencies over a wide range of times. Generally
lengths of treatment will be proportional to the length of the collection and handling process, the
effectiveness of the therapies being applied, and the condition and response of the organ being treated.
In other embodiments, cells can be administered (e.g., by intravenous, intra-arterial, intra-
tracheal, direct injection, etc.) to a donor subject prior to organ harvest. Depending upon the route of
administration, the cells can be administered for a suitable period of time (e.g., minutes to about 1-4
hours, about 4-8 hours, about 8-12 hours, about 12-16 hours, about 16-20 hours, about 20-24 hours). The
organ can be harvested after the period of time by conventional surgical technique(s). After harvest, cells
are contacted (e.g., immediately contacted) with the organ for a time sufficient to allow the cells to
distribute throughout the organ (e.g., less than about 1 hour, about 1-2 hours, about 2-3 hours, about 3-4
hours). Cells can be contacted with the organ by infusion and/or immersion of the organ into a bath
containing the cells. Next, the organ can be stored on ice and/or attached to a reperfusion system,
whereafter the organ is delivered to the transplantation site.
Upon arrival at the transplantation site, the organ can be placed on a reperfusion system (if it
has not been done so already) containing the cells. The organ can then be infused with the cells for a
period of time (e.g., less than about an hour, about 1-2 hours, about 2-3 hours, about 3-4 hours) depending
upon the organ and the reperfusion system. During transplantation, cells can be contacted with the organ
by infusion into the recipient (e.g., intravenously), direct injection into the organ, and/or direct application
to a surface of the organ. Infusion can be done, for example, prior to closure of the vessel(s) entering and
exiting the organ. During liver transplantation, for instance, cells may be infused into the hepatic portal
vein prior to attachment of the vein to the transplanted liver. Additionally or alternatively, cells can be
delivered to the organ after transplantation (e.g., after closure of the vessel(s) entering and exiting the
organ) for a suitable period of time (e.g., minutes to about 1-4 hours, about 4-8 hours, about 8-12 hours,
about 12-16 hours, about 16-20 hours, about 20-24 hours) depending upon the delivery system and the
organ.
Compositions
The invention is also directed to cell populations with specific potencies for achieving any of
the effects described herein. As described above, these populations are established by selecting for cells
that have desired potency. These populations are used to make other compositions, for example, a cell
bank comprising populations with specific desired potencies and pharmaceutical compositions containing
a cell population with a specific desired potency.
EXAMPLE
Methods
Lung harvest and ex vivo perfusion
Following research consent obtained by the local Organ Procurement Agency, LifeGift, the
discarded donated lungs were procured for this study under an established IRB protocol at the Houston
Methodist (IRB(2)1111-0205). Lungs from each of the five patients were procured in a standard fashion
with antegrade Perfadex (Vitrolife AB, Gothenburg, Sweden) 60 ml/Kg flush plus retrograde Perfadex
perfusion through the pulmonary veins. The lungs were then stored in plastic bags containing 1 liter of
Perfadex and were kept on ice during transport. Once the lungs arrived at the Houston Methodist, they
were then stored in a refrigerator @ 4° C for a total 8 hours of cold static storage in order to induce cold
ischemic injury.
Ex Vivo Lung Perfusion (EVLP) was performed with the CE-marked Vivoline LS1 (Vivoline
Medical AB, Lund, Sweden) (Fig. 1) (Wierup, P. et al., Ann Thorac Surg 2006;81(2):460-6;
Ingemansson, R. et al., Ann Thorac Surg 2009;87(1):255-60; and Cypel, M. et al., N Engl J Med
2011;364(15):1431-40). The system was primed with 2.5 L of Steen Solution (XVIVO Perfusion). The
use of washed red blood cell or blood was avoided in order to decrease the number of variables in the
feasibility study. Meropenem 100 mg (AstraZeneca AB, Sodertalje, Sweden) and 10,000 U of Heparin
(LEO Pharmaceutical, Copenhagen, Denmark) were added to the perfusate. Before the lungs were
connected to the EVLP unit, the pH in the solution was corrected to between 7.35 and 7.45 using
trometamol (Addex-THAM, Fresenius Kabi AB, Uppsala, Sweden). In one case where the heart was
procured as well, a Dacron Graft was sutured to the divided pulmonary artery branches in order to
reconstitute the integrity of the pulmonary artery (PA) and facilitate the connection of the lung to the
EVLP circuit. The trachea was connected to the mechanical ventilator via a silicon tube size matching the
tracheal diameter. A temperature probe was positioned inside the left atrium. Initially, for de-airing the
circuit, the lungs were perfused at a flow rate of 0.5 L/min. The shunt for de-airing on the inflow cannula
was kept open until the organ reached 32 ºC and then closed for the rest of the session. The flow was
then increased to 100% of estimated cardiac output for the specific set of lungs. The lungs were then
warmed over 30 minutes to a target of 36ºC and the temperature difference between lung blood inflow
and outflow was not allowed to exceed 8ºC. The flow rate was then increased gradually to a target level
of 70 mL/min per kilogram donor weight, during which the PA pressure was measured continuously and
limited to 15 mm Hg. Rewarming was achieved within 20-30 minutes. When the perfusate temperature
reached 32ºC, mechanical ventilation was started in volume-controlled mode at an initial tidal-volume of
3 ml per kilogram of donor weight with a positive end-expiratory pressure (PEEP) level of 5 cm H O, a
rate of 7-10 breaths/min, and a FiO2 of 0.5. Tidal volume was then increased gradually to a maximum of
7 mL per kilogram of donor weight. Perfusate samples for blood gas analyses were drawn from the
dedicated port of the system.
Cells
Human bone marrow derived MAPCs (Human MultiStem®, Athersys Inc., Cleveland) were
isolated from a single bone marrow aspirate, obtained with consent from a healthy donor, and processed
according to previously described methods (Penn, MS et al., Circ Res 2012;110(2):304-11; Maziarz, RT
et al., Biology of Blood and Marrow Transplantation 2012;18(2 Sup):S264-S265; and clinicaltrials.gov
#NCT01436487, #NCT01240915 and #NCT01841632). In brief, MAPCs were cultured in fibronectin-
coated plastic tissue culture flasks under low oxygen tension in a humidified atmosphere of 5% CO .
Cells were cultured in MAPC culture media (low-glucose DMEM [Life Technologies Invitrogen]
supplemented with FBS (Atlas Biologicals, Fort Collins, CO), ITS liquid media supplement [Sigma],
MCDB [Sigma], platelet-derived growth factor (R&D Systems, Minneapolis, MN), epidermal growth
factor (R&D Systems), dexamethasone ([Sigma], penicillin/streptomycin [Life Technologies Invitrogen],
2-Phospho-L-ascorbic acid [Sigma, St. Louis, MO), and linoleic acid-albumin (Sigma). Cells were
passaged every 3-4 d, harvested using trypsin/EDTA (Life Technologies Invitrogen, Carlsbad, CA). The
cells were positive for CD49c and CD90 and negative for MHC class II and CD45 (all Abs were from BD
Biosciences, Franklin Lakes, NJ). Cells were subsequently frozen at population doubling 30-35 in
cryovials in the vapor phase of liquid nitrogen at a concentration of 1-10 x 10 in 1 ml (PlasmaLyte, 5%
HSA and 10% DMSO). Immediately prior to their use, MAPCs were thawed and used directly.
Cell inoculations, lung incubations, and bronchoalveolar lavage (BAL) fluid and tissue analyses
When the temperature as measured by the intra-atrial probe reached approximately 32ºC,
MultiStem 1 ml vials were thawed, diluted into 19 ml of sterile saline and administered by bronchoscope
into the proximal portion of the LLL bronchus. A similar volume of vehicle (20 ml of sterile saline) was
similarly inoculated into the proximal portion of the RLL bronchus. Five minutes after delivery of
MultiStem, the lungs were connected to a Hamilton-C2 mechanical ventilator. After either 2 or 4 hours
of perfusion on the Vivoline system the experiments were stopped. Five minutes before stopping the
perfusion, the same subsegments of the RLL and LLL that had been previously inoculated with either
cells or vehicle were lavaged with 60 mL saline. The recovered BAL fluid was then separated into
aliquots of either raw BAL fluid for assessing total cell counts and cell differentials or was centrifuged
(1200g x 10 min at 4ºC) and the supernatant was collected in separate tubes, snap frozen, and stored at -
70ºC (Lathrop, MJ et al., Stem Cells Translational Medicine (in press); and Goodwin, M. et al., Stem
Cells 2011:29(7):1137-48). For one lung, BAL fluid samples were also obtained during rewarming phase
before ventilation was started, just prior to MAPC or vehicle delivery.
Total BAL fluid cell numbers were determined using an ADVIA® Hematology Analyzer
(Siemens Diagnostics, Johnson City, TN). Cytospins were made using 5x10 cells centrifuged onto pre-
cleaned, pre-treated glass slides (Corning Incorporate, Corning, NY) at 800 rpm for 8 min, dried
overnight, and stained using DiffQuick (Hema 3 Stain Set, Fisher Scientific, Pittsburgh, PA). Cell
populations were determined by blinded manual count of 200 cells performed by three separate
individuals (Lathrop, MJ et al., Stem Cells Translational Medicine (in press); and Goodwin, M. et al.,
Stem Cells 2011:29(7):1137-48). Protein content in undiluted BAL fluid was assessed by Bradford assay
(Bio-Rad, Hercules, CA). The Human Cytokine Array Kit, Panel A (R&D Systems, Minneapolis, MN)
was used to examine BAL fluid supernatants for soluble cytokines, chemokines, and other substances
including C5/Ca, CD40L, CD54, CXCL1, CXCL10, G-CSF, Gro-1α, IL-1α, IL-1β, IL-1RA, IL-6, IL-8,
IL-10, IL-16, IL-23, IP-10, I-TAC, MCP-1, MIF, PAI-1, RANES, serpin E1, sICAM, sTREM-1, TNFα,
and the relative amount of cytokine compared to internal controls determined on a UVP Bioimaging
system.(Upland, CA). Elisas for other specific cytokines were performed according to manufacturer’s
instructions, IL-10 (R&D Systems, Minneapolis, MN, Cat#:D1000B), and STC1, TSG-6 and iNOS
(MyBioSource, San Diego, CA, Cat#s: MBS946255, MBS926793, MBS723617).
Histological assessments
Following BAL at the end of the perfusion period, the lungs were subsequently gravity fixed
with 10% formalin at room temperature for 1 hour. Fixed lungs were dissected and the areas where cells
were instilled stored in 10% formalin prior to paraffin fixation. Mounted 5 µm sections were then
evaluated for histologic appearance. Lung inflammation was scored on 10 airways per animal, in a
blinded fashion by three individuals, based on the presence and intensity of peri-bronchial cell infiltrates
compared to known positive and negative controls using an established semi-quantitative scoring system,
using a 0-3 range and 0.5 scale increments as previously described (Lathrop, MJ et al., Stem Cells
Translational Medicine (in press); and Goodwin, M. et al., Stem Cells 2011:29(7):1137-48).
qPCR analyses of tissue inflammatory markers
Lung biopsy samples from lungs 2-5 were obtained using an automatic stapler (Covidien
GIA™ DST Series™ 80mm) from the periphery of the LLL and RLL just prior to cell or vehicle infusion
at 2 and 4 hours after cell or vehicle infusion and at the end of the experiment. The samples were snap
frozen and subsequently homogenized and the expression levels of inflammatory cytokine mRNAs
determined by qPCR (see details below).
Samples were homogenized in RNA lysis buffer and RNA extracted using the RNeasy kit
(Qiagen, Germantown, MD) according to manufacturer’s instructions. Additional DNase treatment was
performed using the DNA-free kit (Life Technologies, Carlsbad, CA). RNA concentration was measured
by NanoDrop 2000 (Thermo Scientific, Waltham, MA) and 1 µg RNA was reverse transcribed using M-
MLV Reverse Transcriptase (Promega, Madison, WI) followed by RNAse treatment using RNace-it
Cocktail (Agilent, Santa Clara, CA). Reverse transcriptase negative samples and water were run as
controls. 5 µl of the cDNA was mixed with SYBR green (Promega) and primers (IDT) and run on the
ABI 7500 FAST system (Applied Biosystems, Foster City, CA). The samples were normalized to
GAPDH and expressed as a percent of Human Reference (Agilent) +/- standard deviation.
Primer sequences were as follows:
VEGFA–F1 (SEQ ID NO:1);
VEGFA–R1 (SEQ ID NO:2);
IGF1–F4 (SEQ ID NO:3);
IGF1–R4 (SEQ ID NO:4);
EGF–F1 (SEQ ID NO:5);
EGF-R1 (SEQ ID NO:6);
IL-10–F2 (SEQ ID NO:7);
IL-10–R2 (SEQ ID NO:8);
FGF2–F1 (SEQ ID NO:9);
FGF2–R1 (SEQ ID NO:10);
HGF–F1 (SEQ ID NO:11);
HGF–R1 (SEQ ID NO:12);
CCL5–F1 (SEQ ID NO:13);
CCL5–R1 (SEQ ID NO:14);
TGFB1–F1 (SEQ ID NO:15);
TGFB1–R1 (SEQ ID NO:16)
CXCL10–F1 (SEQ ID NO:17);
CXCL10–R1 (SEQ ID NO:18);
NOS3–F2 (SEQ ID NO:19);
NOS3–R2 (SEQ ID NO:20);
STC1–F1 (SEQ ID NO:21);
STC1–R1 (SEQ ID NO:22);
GAPDH–F1 (SEQ ID NO:23);
GAPDH–R1 (SEQ ID NO:24);
ANGPT1–F2 (SEQ ID NO:25);
ANGPT1–R2 (SEQ ID NO:26);
NOS2–F1 (SEQ ID NO:27);
NOS2–R1 (SEQ ID NO:28);
TNFAIP6–F1 (SEQ ID NO:29);
TNFAIP6–R1 (SEQ ID NO:30);
FGF7–F1 (SEQ ID NO:31); and
FGF7–R1 (SEQ ID NO:32).
Statistical analysis
Groups were compared using either one way or two-way ANOVA with a Fishers LSD post-test
or by direct analysis between two groups by Student’s T-test, using a Welch’s correction for unequal
variances, as appropriate (Lathrop, MJ et al., Stem Cells Translational Medicine (in press); and Goodwin,
M. et al., Stem Cells 2011:29(7):1137-48).
Results
The relevant clinical characteristics of the donor lungs are summarized in Table 1.
Table 1: Clinical Characteristics of the Donor Lungs
Donor
1 2 3 4 5 Mean + SD
Characteristics
Age 55 56 44 66 50 54.2 + 3.6
Sex Male Male Male Female Male
Cause of Death CVA SH Asphyxiation IH MVA
236.4 +
PaO @100% FiO 150 186 254 443 149
55.1
Peep 10 10 10 5 10 9.0 + 1.0
Edema-Right
lower lobe
Infiltrate- Infiltrate- Clear
Radiographic Findings Infiltrate-Edema
collapse,
Edema Edema
Right pleural
effusion
Lung Appearance Edematous
Edematous Edematous Multiple
Contusions,
surface
Edematous
nodules
CVA:Cerebrovascular accident; SH:Subarachnoid hemorrhage; IH:Intracranial Hemorrhage; MVA:
motor vehicle accident.
Donor age ranged from 44-66 and three of the five donor lungs were obtained from patients
with devastating neurologic events, one from asphyxia, and one from a motor vehicle accident. Four of
the five lungs were not deemed suitable for transplant because of poor functional status including low
PaO values with a mean of 184.75 mmHg at 100% FiO at + a PEEP of 10 mmHg. These lungs also had
radiographic abnormalities, variously including contusions, significant emphysema, or lobar collapse that
did not respond to recruitment maneuvers in the operating room. Each of these lungs also had
radiographic signs of pulmonary edema with two having also pleural effusion and all were noted to be
variably edematous following surgical removal. Lung #5 had RLL collapse on CXR but expanded
following removal and bronchoscopic removal of mucus plugs. One lung (lung #4) was physiologically
suitable for donation with normal appearance, clear CXR, and good oxygenation on 5 mmHg PEEP but
not utilized due to the presence of small surface nodules that were subsequently found to be benign on
biopsy.
A summary of the protocol utilized for each lung is presented in Table 2 and also in schematic
form in Fig. 1.
Table 2: Summary of Experimental Protocol
Donor Lung 1 2 3 4 Mean + SD
Duration of
8 8 8 8 8.0 + 0
Cold Static
Storage (hours)
Rewarming
22 25 28 26 25.0 + 1.0
Time
(minutes)
Duration of Ex
4 2.5 4 4 3.6 + 0.6
Vivo Perfusion
(hours)
7 7 6
MAPC to 10 MAPC to 10 MAPC to
MAPC to LLL 10 MAPC to LLL
LLL LLL LLL
Cells or Vehicle
Vehicle to RLL Vehicle to RLL
Delivered
Vehicle to RLL Vehicle to RLL Vehicle to RLL
Overall the lungs had similar cold storage (8 hrs) and rewarming (25 + 2.2 minutes) times and
subsequently similar reperfusion times (3.7 + 0.6 hours) following bronchoscopic administration of cells
or vehicle. At the end of the reperfusion period, there was some degree of further edema that had
developed in each lung and lung number 4 had also newly developed some degree of edema. However,
overall there was less visible edema and inflammation in the MAPC-treated (LLL) vs. vehicle-treated
(RLL) lobes, even with the lower dose of MAPCs utilized in lung #4. Representative images are shown
in Fig. 2.
Histologic assessment of the lungs at the end of the reperfusion period demonstrated that
although patches of inflamed areas could be found in some of the MAPC-treated LLLs, there was
significantly less overall inflammation in 4 out of the 5 lungs and also averaged over all 5 lungs, as
assessed by semi-quantitative scoring of peribronchial, perivascular, and alveolar septa edema and by
presence of inflammatory cell infiltrates (Fig. 3). Representative photomicrographs are depicted in Fig. 4.
Total BAL fluid cell counts were obtained in two out of four lungs (lungs 3 and 5) receiving
the higher cell dose. In both cases, there was a significant decrease in the MAPC-treated LLL compared
to the vehicle-treated RLL (Fig. 5A). A trend towards decrease in total BAL fluid cell counts was also
observed in the lung receiving the lower MAPC dose (lung 4, Fig. 5A). Cell differentials obtained on
BAL fluid samples from all five lungs demonstrated a consistent increase in neutrophils and eosinophils
in the vehicle-treated RLL that was ameliorated in the MAPC-treated LLL (Fig. 5B). Measurements of
BAL fluid total protein levels was variable between the lungs but a consistent decrease in total protein in
the MAPC-treated LLL vs. vehicle-treated RLL was observed in all 5 lungs (Fig. 5C).
An increase in levels of IL-10 in the MAPC-treated LLL compared the vehicle treated RLL
was observed (Fig. 6). However, other soluble anti-inflammatory mediators implicated in pre-clinical
models of MAPC actions in lung injury and other models, such as IL-1RA, STC, TGS-6, and iNOS, were
not reliably increased in the MAPC-treated LLL in any of the 5 lungs (Fig. 6). Tissue mRNA levels were
assessed in 4 of 5 lungs (lungs 2-5) by qPCR analyses of biopsy samples obtained prior to cell or vehicle
administration and then after either 2 or 4 hours of reperfusion period. Overall, patterns of tissue mRNA
levels were more consistent between the 4 lungs. Comparable to BAL fluid levels of IL-10 protein, there
was a 3.5-fold increase in the levels of tissue IL-10 mRNA in the MAPC-treated LLL compared to only a
1.6-fold increase in vehicle-treated RLL as assessed at 2hrs (Fig. 7). Similar increases in LLL vs. RLL
were also observed at 2 hrs in mRNA levels of Angpt1 and STC1. Interestingly, for both the LLL and
RLL there was a large increase in the fold expression of TSG6 from 2 to 4 hours.
Discussion
A number of different methods have been studied to improve the viability of donor lungs and to
decrease either warm or cold ischemic inflammatory injury. These include a flushing solution with
extracellular characteristics delivered both in an antegrade and retrograde fashion and the use of a
portable ex vivo preservation system currently under clinical investigation for use in transport of donor
lungs (Machuca, TN et al., Surg Clin North Am 2013;93(6):1373-94). Different areas of research for
therapeutic interventions aim to modulate the response induced by ischemia and reperfusion. For example
experimental animal models have shown beneficial effect from gene therapy deliver of IL-10 (Cypel, M.
et al., Sci Transl Med. 2009;1(4):4-9) and from adenosine receptor activation (Fernandez, LG et al., J
Thorac Cardiovasc Surg 2013;145(6):1654-9; and Mulloy, DP et al., Ann Thorac Surg 2013;95(5):1762-
7). However, while the experimental data are promising, it is unlikely that modulating one out of many
inflammatory pathways can regulate a phenomenon that alters several cellular mechanisms involved, as
innate and adaptive immunity, the activation of the complement cascade, endothelial dysfunction, and the
triggering of cell death. In contrast, bone marrow-derived MSCs and MAPCs have the unique potential
of acting on multiple inflammatory pathways involved in ischemia/reperfusion injury.
Ex vivo lung perfusion (EVLP) was originally designed as a method to assess the quality of
lungs from donation after cardiac death (DCD) and from other non-acceptable donor lungs (Wierup, P. et
al., Ann Thorac Surg 2006;81(2):460-6; and Ingemansson, R. et al., Ann Thorac Surg 2009;87(1):255-
60). This technique is currently under clinical trial for the evaluation and reconditioning of potential
donor lungs that under current criteria are not deemed suitable for transplant (Cypel, M. et al., N Engl J
Med 2011;364(15):1431-40). EVLP further offers an opportunity to administer MSCs or MAPCs
directly into the donor lung by either intratracheal or intravascular routes prior to implantation. Using this
approach the inventors chose to initially assess direct airway MAPC administration into a single lobe with
the contralateral lung as comparison to directly assess effects within each individual lung. The cold
ischemic storage (8 hours of total cold storage) was prolonged beyond the actual times generally accepted
for the lungs to potentiate any IRI that might develop and thus to maximize potential anti-inflammatory
actions of the MAPCs. The inventors also chose to use “off the shelf” non-HLA matched MAPCs as
proof of feasibility. Moreover, the inventors demonstrate a consistent and potent anti-inflammatory effect
of the MAPCs. Notably, the change in cytokine profile, particularly increase in IL-10, may be
particularly beneficial for IRI.
From the above description of the present invention, those skilled in the art will perceive
improvements, changes and modifications. Such improvements, changes, and modifications are within
the skill of those in the art and are intended to be covered by the appended claims. All patents, patent
applications, and publications cited herein are incorporated by reference in their entirety.
Claims (19)
1. An ex vivo method of treating an organ or tissue for transplantation, comprising perfusing the organ or tissue with exogenous stem cells, wherein the stem cells are multipotent adult progenitor cells (MAPCs) characterized in that they are non-embryonic, non-germ cells that express telomerase, have a normal karyotype, are not tumorigenic, and wherein the stem cells have undergone at least 10-40 cell doublings in cell culture prior to their perfusion into the organ or tissue, wherein the organ or tissue is perfused with the stem cells prior to transplantation into a recipient.
2. Exogenous stem cells, when used for an ex vivo method of treating an organ or tissue for transplantation, comprising perfusing the organ or tissue with the exogenous stem cells, wherein the stem cells are MAPCs characterized in that they are non-embryonic, non-germ cells that express telomerase, have a normal karyotype, are not tumorigenic, and wherein the stem cells have undergone at least 10-40 cell doublings in cell culture prior to their perfusion into the organ or tissue, wherein the organ or tissue is perfused with the stem cells prior to transplantation into a recipient.
3. The method of claim 1, or the exogenous stem cells when used for an ex vivo method of claim 2, wherein the stem cells can differentiate into cell types of at least two of endodermal, ectodermal, and mesodermal germ layers.
4. The method of claim 1, or the exogenous stem cells when used for an ex vivo method of claim 2, wherein the stem cells express oct4.
5. The method of claim 3, or the exogenous stem cells when used for an ex vivo method of claim 3, wherein the stem cells can differentiate into cell types of endodermal, ectodermal, and mesodermal germ layers.
6. The method of claim 1, or the exogenous stem cells when used for an ex vivo method of claim 2, wherein the stem cells are non-embryonic, non-germ cells that express oct4 and telomerase and can differentiate into cell types of at least two of endodermal, ectodermal, and mesodermal germ layers.
7. The method of claim 6, or the exogenous stem cells when used for an ex vivo method of claim 6, wherein the stem cells can differentiate into cell types of endodermal, ectodermal, and mesodermal germ layers.
8. The method of any one of claims 1 and 3 to 7, or the exogenous stem cells when used for an ex vivo method of claim 2 to 7, wherein the stem cells are non-HLA matched, allogeneic cells.
9. The method of any one of claims 1 and 3 to 8, or the exogenous stem cells when used for an ex vivo method of claim 2 to 8, wherein the stem cells have undergone at least 30-35 cell doublings.
10. The method of any one of claims 1 and 3-9, or the exogenous stem cells when used for an ex vivo method of claim 2 to 9, wherein the concentration of stem cells perfused into the organ or tissue is about 1 x 10 cells/ml to about 10 x 10 cells/ml.
11. The method of any one of claims 1 and 3 to 10, or the exogenous stem cells when used for an ex vivo method of claim 2 to 10, wherein the stem cells are perfused into the organ or tissue for about 2-4 hours.
12. The method of any one of claims 1 and 3 to 11, or the exogenous stem cells when used for an ex vivo method of claim 2 to 11, wherein the stem cells are contained in a fluid for perfusion into the organ or tissue or in a carrier for intra-organ or intra-tissue administration.
13. The method of any one of claims 1 and 3 to 12, or the exogenous stem cells when used for an ex vivo method of claim 2 to 12, wherein the stem cells are contained in a medium in which the organ or tissue is bathed prior to transplantation.
14. The method of any one of claims 1 and 3 to 13, or the exogenous stem cells when used for an ex vivo method of claim 2 to 13, wherein the organ or tissue is selected from the group consisting of lung, kidney, heart, liver, pancreas, thymus, gastrointestinal tract, and composite allografts.
15. The method of any one of claims 1 and 3 to 14, or the exogenous stem cells when used for an ex vivo method of claim 2 to 14, wherein perfusion with the stem cells reduces inflammation in the organ or tissue.
16. The method of any one of claims 1 and 3 to 15, or the exogenous stem cells when used for an ex vivo method of claim 2 to 15, wherein perfusion with the stem cells reduces the occurrence of inflammatory cells in the organ or tissue.
17. The method of any one of claims 1 and 3 to 16, or the exogenous stem cells when used for an ex vivo method of claim 2 to 16, wherein perfusion with the stem cells reduces inflammatory cytokines in the organ or tissue.
18. The method of any one of claims 1 and 3 to 14, or the exogenous stem cells when used for an ex vivo method of claim 2 to 14, wherein perfusion with the stem cells reduces ischemic-reperfusion injury.
19. The method of any one of claims 1 and 3 to 18, or the exogenous stem cells when used for an ex vivo method of claim 2 to 18, wherein the stem cells are derived from human bone marrow.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201361811525P | 2013-04-12 | 2013-04-12 | |
US61/811,525 | 2013-04-12 | ||
PCT/US2014/034015 WO2014169277A1 (en) | 2013-04-12 | 2014-04-14 | Improving organs for transplantation |
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Publication Number | Publication Date |
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NZ714064A NZ714064A (en) | 2021-10-29 |
NZ714064B2 true NZ714064B2 (en) | 2022-03-01 |
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