U.S. patent application number 14/554932 was filed with the patent office on 2015-09-03 for immune privileged and modulatory progenitor cells.
The applicant listed for this patent is Dolores BAKSH, John E. DAVIES, Jane E. ENNIS, Alejandro GOMEZ-ARISTIZABAL. Invention is credited to Dolores BAKSH, John E. DAVIES, Jane E. ENNIS, Alejandro GOMEZ-ARISTIZABAL.
Application Number | 20150246079 14/554932 |
Document ID | / |
Family ID | 38667370 |
Filed Date | 2015-09-03 |
United States Patent
Application |
20150246079 |
Kind Code |
A1 |
DAVIES; John E. ; et
al. |
September 3, 2015 |
IMMUNE PRIVILEGED AND MODULATORY PROGENITOR CELLS
Abstract
Described herein is a method for modulating an immune reaction
between lymphocytes and a body recognized by the lymphocytes as
foreign. The method exploits the immunomodulating activity of a new
class of progenitor cells termed HUCPVCs derived from the
perivascular region of human umbilical cord. The method can also
employ soluble factors exuded by cultured HUCPVCs. The method is
useful to treat immune disorders including graft versus host
disease, autoimmune disorders, and the like.
Inventors: |
DAVIES; John E.; (Toronto,
CA) ; ENNIS; Jane E.; (Oakville, CA) ;
GOMEZ-ARISTIZABAL; Alejandro; (Toronto, CA) ; BAKSH;
Dolores; (Mississauga, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DAVIES; John E.
ENNIS; Jane E.
GOMEZ-ARISTIZABAL; Alejandro
BAKSH; Dolores |
Toronto
Oakville
Toronto
Mississauga |
|
CA
CA
CA
CA |
|
|
Family ID: |
38667370 |
Appl. No.: |
14/554932 |
Filed: |
November 26, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13630312 |
Sep 28, 2012 |
8900573 |
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14554932 |
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12299736 |
Jul 8, 2009 |
8277794 |
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PCT/CA2007/000781 |
May 4, 2007 |
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13630312 |
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60746500 |
May 5, 2006 |
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Current U.S.
Class: |
424/184.1 |
Current CPC
Class: |
C12N 5/0665 20130101;
A61K 35/44 20130101; A61P 37/00 20180101; A61P 37/02 20180101; A61P
37/06 20180101; A61P 17/00 20180101; A61K 35/12 20130101; A61K
35/51 20130101; A61P 35/02 20180101 |
International
Class: |
A61K 35/51 20060101
A61K035/51 |
Claims
1. A method for modulating an immune reaction between lymphocytes
and a body recognized by the lymphocytes as foreign, comprising the
step of introducing, in an amount effective to inhibit or reduce
said immune reaction, an agent selected from (1) umbilical cord
perivascular cells, and/or (2) an immunomodulating soluble factor
produced by said cells.
2. The method according to claim 1, wherein said method comprises
administering said agent to a subject having or at risk of
developing an adverse immune reaction.
3. The method according to claim 2, wherein the subject has or is
at risk for graft versus host disease.
4. The method according to claim 2, wherein the subject has or is
at risk for a mixed lymphocyte reaction.
5. The method according to claim 2, wherein the subject has or is
at risk for graft rejection.
6. The method according to claim 5, wherein the graft is a skin
graft.
7. The method according to claim 5, wherein the graft is an organ
graft.
8. The method according to claim 5, wherein the graft is a marrow
graft.
9. The method according to claim 5, wherein the graft is a
peripheral blood graft.
10. The method according to claim 2, wherein the subject has an
autoimmune disorder.
11. The method according to claim 2, wherein the subject is
afflicted with a leukemia and is at risk for graft versus host
disease.
12. The method according to claim 1, wherein said method comprises
exposing a tissue graft, prior to transplantation thereof into a
graft recipient, to said agent, wherein said exposing reduces graft
versus host disease in said graft recipient.
13. The method according to claim 1, wherein the agent is umbilical
cord perivascular cells.
14. The method according to claim 13, wherein the agent is human
umbilical cord perivascular cells (HUCPVCs).
15. The method according to claim 1 wherein the umbilical cord
vascular cells comprise a transgene that encodes a protein of
interest.
16. The method according to claim 1, wherein the umbilical cord
perivascular cells are substantially MHC double negative
HUCPVCs.
17. The method according to claim 2, wherein the HUCPVCs are
administered in a unit dose in the range from 0.01 to 5 million
HUCPVC cells per kilogram of said subject.
18. The method according to claim 1, wherein the agent is an
immunomodulating soluble factor produced by HUCPVCs.
19. The method according to claim 18, wherein the immunomodulating
soluble factor is provided as an extract of medium conditioned by
HUCPVC growth.
20. An extract comprising an immunomodulating soluble factor
produced upon culturing of HUCPVs.
Description
FIELD OF THE INVENTION
[0001] This invention relates to progenitor cells that are
immunoprivileged and/or immunomodulatory, their production, their
formulation, and their therapeutic use.
BACKGROUND TO THE INVENTION
[0002] Adult bone marrow (BM) is the most common source of
mesenchymal stem/progenitor cells (MSCs), (also called Mesenchymal
Stromal Cells.sup.1) which are functionally defined by their
capability of differentiating into the skeletal tissues:
bone.sup.2-4, cartilage.sup.5-7, fat.sup.8 and muscle.sup.9 in
vitro. MSCs are classically distinguished from the heterogeneous
milieu of cells through adhesion to tissue culture plastic and the
formation of colony unit-fibroblasts (CFU-Fs), the frequency of
which are 1:100,000-1:500,000 nucleated cells in adult
marrow.sup.10, and studies have now identified a suite of markers
with which MSCs are categorized.sup.10,11. This low proportion of
MSCs leads to the necessity of culture expansion and selection
before use to attain the appropriate cell numbers for any kind of
cellular therapy. There are other emerging sources of MSCs such as:
adipose tissue.sup.12, trabecular bone.sup.13 and fetal
liver.sup.14 which have a CFU-F frequency of: 1:32.sup.15,
1:636.sup.13 and 1:88,495.sup.14 respectively. While adipose tissue
does appear to have the highest frequency of progenitors, the
doubling time of those cells ranges between 3.6 to 4.4 days.sup.15,
and the extraction procedure is complicated, invasive, and
lengthy.sup.12. Harvesting trabecular bone results in low cell
yield (89.times.10.sup.6 cells/gram of bone from young
donors.sup.13), especially when combined with the CFU-F frequency;
and is extremely invasive resulting in donor site morbidity.
[0003] Unique among these new sources of MSCs are human umbilical
cord perivascular cells (HUCPVCs), which are an easily accessible,
highly proliferative source of cells with a population doubling
time of 20 hours (dependent on serum).sup.16. The frequency of
CFU-Fs in HUCPVCs is 1:300 at passage 0 but increases to 1:3 at
passage 1.sup.17, which is orders of magnitude higher than bone
marrow.sup.16. Therefore HUCPVCs represent a population of cells
with an extremely high proportion of MSCs which proceed to divide
very quickly, thus making them an excellent candidate for clinical
mesenchymal therapies. These cells have been used in various assays
to determine their marker expression phenotype and differentiation
potential.sup.16, 18, and have been found to be either
bioequivalent to, or perform better than, BM-MSCs.
[0004] In addition to their ability to differentiate, MSCs also
have potential immunological uses as BM-MSCs have been shown to be
both immunoprivileged and immunomodulatory.sup.19-21. These terms
refer to a cell's ability to evade recognition from a mismatched
host's immune system, and the ability to mitigate an ongoing
response by that system, respectively. MSCs from several sources
other than bone marrow have been tested for their immunogenicity in
in vitro cultures. MSCs from adipose tissue derived from adult
dermolipectomies were shown to be capable of both immunoprivilege
and immunomodulation in vitro.sup.22, whereas fetal liver cells
were found to be capable of avoiding a mismatched immune response,
however they were not able to modulate alloreactivity caused by two
mismatched populations of lymphocytes.sup.23, 24. Thus, the source
of MSCs directly affects those cells' immunogenic capabilities.
[0005] This in vitro work has begun to be validated in the clinical
setting; for example, a boy was rescued from severe acute graft vs.
host disease (GvHD) by transfusion of haploidentical bone marrow
MSCs from his mother.sup.25. One year post treatment, in comparison
to a cohort of patients suffering from the same level of severity
of the disease, he was the only one alive. Since this initial
patient, a suite of 8 patients have been treated with BM-MSCs, of
which 6 showed a complete remission of symptoms.sup.26. Allogeneic
BM-MSCs have also been used in Crohn's Disease to treat patients
who are refractory to current treatments, and this treatment is
currently in clinical trials in the United States.sup.27, 28. Fetal
liver MSCs have shown efficacy in the early treatment of
osteogenesis imperfecta (OI). MSCs from a male fetal liver were
transplanted into an unrelated 32 week female fetus with severe OI,
who had suffered several intrauterine fractures.sup.29. Following
the transplantation, the remainder of the pregnancy proceeded
normally, and there were no further fractures. This patient has
been followed up to 2 years after birth, and the child has shown a
normal growth curve and has suffered only 3 fractures. Using an
XY-specific probe, the patient was found to have 0.3% engraftment
in a bone biopsy.
[0006] In addition to undifferentiated cells, osteogenically
induced rabbit BM-MSCs were found to be immunoprivileged and
immunomodulatory in vitro, but when transplanted in vivo the
immunomodulatory capacity was lost.sup.30. This would not affect
the function of the cells however; as they only require protection
from an immune response in order to fulfill their role. In a more
involved induction, murine bone marrow MSCs were manipulated to
release erythropoietin and implanted in mice, which resulted in
significantly less engraftment compared to un-manipulated
controls.sup.31. Thus, manipulation of MSCs can lead to their loss
of immunomodulation and/or immunoprivilege and can be crucial to
the survival and function of the graft.
[0007] There is evidence to support that the immunoprivilege of
MSCs transcends species barriers, and they can be used
xenogeneically. This was first demonstrated by Bartholomew et al
who used human BM-MSCs in baboons, and showed enhanced skin graft
survival.sup.21. While the end result of this study was positive,
the specific fate of the administered cells was not determined.
Wang et al. have utilized GFP transfected cells and histological
analyses to studied the survival of xenogeneic BM-MSCs, and showed
that the cells survive up to the 11 week timepoint without
immunosuppression, however there was an increased host immune
reaction.sup.32. MSCs have also been reported to survive in
xenogeneic transplantations in two cardiac models.sup.33, 34. In
preliminary work with HUCPVCs, the cells were delivered
peritoneally in permeable chambers. After 3 weeks, there was no
noticeable inflammation noted upon macroscopic
visualization.sup.35. This is encouraging preliminary work
indicating the potential for not only the immunoprivilege of
HUCPVCs, but also for their ability to test them in animal models
without rejection.
[0008] The inventors investigated the immunoprivileged and
immunomodulatory properties of HUCPVCs in vitro by conducting both:
co-cultures of HUCPVCs with unmatched lymphocytes, and mixed
lymphocyte cultures (MLCs) populated by two HLA mismatched donors.
Also studied were HUCPVC death, lymphocyte proliferation and
activation with varying levels of HUCPVCs present in both naive and
activated lymphocyte environments. In addition, the necessity for
cell contact for the observation of immunological effects was
investigated.
SUMMARY OF THE INVENTION
[0009] The inventors now report herein a series of experiments
which illustrate both the immunoprivileged and immunomodulatory
capabilities of HUCPVCs when tested in one and two-way in vitro
mixed lymphocyte cultures (MLCs). Additionally, MLCs were performed
which reveal a HUCPVC-induced decrease in activation of previously
stimulated lymphocytes. The inventors further show that the HUCPVC
immunomodulatory function is mediated through a soluble factor(s)
produced upon culturing of the HUCPVCs, as cell contact is not
required for the immunomodulatory effect to be observed.
Furthermore, the inventors illustrate that HUCPVCs are capable of
modulating a two-way in vitro MLC, and describe the use of these
cells for cellular therapy applications, particularly to modulate
the immune response.
[0010] Thus, in one of its aspects, the present invention provides
a method for treating a subject having, or at risk of developing,
an adverse immune reaction, comprising the step of administering to
the subject an immunomodulating effective amount of (1) a cell
population comprising, and preferably consisting essentially of,
human umbilical cord perivascular cells (HUCPVCs), and/or (2) an
immunomodulating soluble factor produced upon culturing of said
cells. In related embodiments, the method is applied to treat
recipients of allogeneic or xenogeneic grafts, including cells,
tissues and organs, to reduce the onset or severity of adverse
immune reaction thereto, including graft versus host disease. In a
general aspect, the present invention thus provides a method for
modulating an immune reaction between lymphocytes, such as
peripheral blood lymphocytes, and a body recognized by the
lymphocytes as foreign, comprising the step of introducing a
formulation comprising a physiologically tolerable vehicle and
HUCPV cells or immunomodulating soluble factors that are
extractable therefrom, in an amount effective to modulate and
particularly to inhibit or reduce that immune reaction.
[0011] In a related aspect, the present invention provides for the
use of HUCPVC cells or an immunomodulating soluble factor produced
thereby in the manufacture of a medicament for the treatment of a
subject having or at risk of developing an adverse immune reaction,
or for the treatment of a graft prior to transplantation, to
mitigate or reduce immune reaction between the graft and
recipient.
[0012] In another of its aspects, the present invention provides a
formulation, in unit dosage form or in multidosage form, comprising
an immunomodulating effective amount of HUCPVCs and/or an
immunomodulating soluble factor produced thereby, and a
physiologically tolerable vehicle therefor.
[0013] In a further aspect, the present invention provides an
immunomodulating extract, or an immunomodulating fraction thereof,
comprising one or more soluble factors produced by cultured
HUCPVCs.
[0014] In another aspect, the present invention provides a
treatment method as described hereinabove, wherein the administered
cells are obtained and administered without cryogenic storage.
[0015] In a further aspect of the present invention, the
administered HUCPVCs are immunoprivileged and immunomodulatory
cells. In embodiments, the HUCPVCs are substantially lacking both
the MHC class I and MHC class II phenotypes. In a related
embodiment, the administered HUCPVCs are obtained by thawing of a
population of frozen HUCPVCs.
[0016] In a further embodiment of the present invention, the
administered immunoprivileged HUCPVCs are engineered genetically,
and incorporate a transgene that encodes a heterologous protein of
interest, particularly but not exclusively including a protein
effective to manage the immune system such as a protein that
enhances immunomodulation, and especially a protein that inhibits
adverse immune reaction, such as CTLA4.
[0017] These and other aspects of the present invention are
described in greater detail below, with reference to the
accompanying Figures, in which:
BRIEF REFERENCE TO THE FIGURES
[0018] FIG. 1: Cell counts of HUCPVCs after 7 days in culture
post-MMC treatment (n=2). The cells were treated with ranging
concentrations of MMC for 20 minutes at 37.degree. C. at 5%
CO.sub.2 and assayed for their proliferation. All are seen to be
significantly lower than control (p<0.001).
[0019] FIG. 2: Proliferation of cells plated in wells treated and
untreated with MMC. Proliferation was measured using flow cytometry
for BrdU, and quantified using mean fluorescence intensity. There
was no significant difference between treated and untreated wells
(p=0.62).
[0020] FIG. 3: HUCPVC death was measured using mean fluorescence
intensity (MFI) for annexin 5, an early cell death marker, after 4
hours of co-incubation with PBLs from Donor 1 (n=5). There was a
significant increase in average annexin 5 expression in the culture
with 10% HUCPVCs relative to control (p=0.01, indicated by *); this
was the only significant increase.
[0021] FIG. 4: Lymphocyte proliferation was measured using mean
fluorescence intensity (MFI) for BrdU, after 6 days of
co-incubation with varying levels of HUCPVCs (n=5). There was a
significant increase in average BrdU expression in the culture with
10% HUCPVCs relative to control (p=0.02, indicated by *).
[0022] FIG. 5: Total lymphocyte cell number was measured from day 1
to 6 across treatments of 10 and 40% HUCPVCs added on day 0, 3 or 5
(n=3). It can be seen that by day 6, the control lymphocytes have
increased in number in response to each other, while the treatments
with HUCPVCs were significantly lower regardless of percentage or
day added (p<0.05%, indicated by *).
[0023] FIG. 6: Total lymphocyte count was measured from day 1 to 6
across treatments of 10 and 40% HUCPVCs added on a TransWell.TM.
insert (n=3). It can be seen that on any day, there is no
significant difference between the allogeneic control and the
HUCPVC treatments.
[0024] FIG. 7: HUCPVCs do not increase resting or activated
lymphocyte cell number. Addition of HUCPVCs showed no significant
increase in lymphocyte cell number compared to controls over 6 days
in culture (n=6). This figure shows the average cell numbers,
+standard deviations.
[0025] FIG. 8: BrdU expression of PBLs in a co-culture with HUCPVCs
measured with flow cytometry. The percentage of cells dividing does
not increase with addition of HUCPVCs, irrespective of dose (n=3).
Control was the BrdU expression of the PBLs without HUCPVCs.
[0026] FIG. 9: HUCPVCs act through a soluble factor. HUCPVCs are
able to significantly reduce lymphocyte cell number in MLCs, when
separated using a TransWell insert. The average control lymphocyte
cell number has been set to 100% in the figure to reduce the
variation in counts between experiments. This figure shows average
percentage lymphocyte cell counts, +standard deviation (n=6).
(p*<0.05)
[0027] FIG. 10: HUCPVCs reduce CD25 expression in co-cultures of
activated lymphocytes. This figure illustrates both the average
percentage expression (bar) and mean fluorescence intensity (line)
of CD25 expression on lymphocytes co-cultured with and without 10%
HUCPVCs. Lymphocytes were stained with PKH26 to ensure proper
detection of the population, and results are gated on PKH26
expression. Averages are .+-.standard deviations (n=3).
(*p<0.05)
[0028] FIG. 11: CD45 expression in a two-way MLC (with activated
lymphocytes) with HUCPVCs. The activated lymphocytes were stained
with PKH26 to delineate them from the inactive population, and
results are gated on PKH26 expression. Both percentage expression
(bar*p<0.05) and MFI (line+p<0.05) are shown (n=3). Control
was the CD45 expression of the ATL with no HUCPVCs.
[0029] FIG. 12A is a photomicrograph showing HUCPVCs transfected
with Green Fluorescent Protein (GFP). These cells were transfected
with a lentiviral vector, using established techniques. FIG. 12B is
a graph showing an expression level of GFP in HUCPVCs of
97.89%.
[0030] FIG. 13A is a graph showing High-throughput Cancer
Pathfinder Gene Array results for bone marrow-derived MSCs. FIG.
13B is a graph showing High-throughput Cancer Pathfinder Gene Array
results for HUCPVCs. Genes in parentheses represent those genes
which are absent.
DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS
[0031] The present invention provides novel and clinically useful
applications of HUCPVCs, particularly in the treatment of
conditions that would benefit from a reduction in the adverse
response elicited by alloantigenic and xenoantigenic bodies,
resulting either from an adverse immune response by the host, or
from an adverse immune reaction by the antigenic body to the host.
More generally, the present invention provides a method in which
HUCPVCs and/or soluble factors produced by them are introduced to
inhibit or reduce immune reactions between lymphocytes and bodies
recognized as foreign.
[0032] As used herein, such bodies can include any living or dead
biological material that is delivered to or invasive to the body of
a mammal, including a human. Antigenic such bodies are those which
in the normal course elicit an immune response either by the
recipient or by the body, for instance where the body itself
comprises immune cells including lymphocytes, such as a bone
marrow, tissue or organ graft. Alloantigenic bodies are bodies that
are antigenic between individuals within the same species;
xenoantigenic bodies are antigenic between individuals of different
species. Autologous bodies are bodies from the recipient. In
embodiments, the bodies are HLA mismatched bodies. In other
embodiments, the bodies comprise HLA mismatched lymphocytes.
[0033] While the mechanism of HUCPVC immunomodulatory action is not
completely understood, it is expected that the HUCPVCs and soluble
factors produced by them have an effect on the major cell
populations involved in alloantigen recognition and elimination,
such as antigen presenting cells, T cells including cytotoxic T
cells, and natural killer cells.
[0034] The HUCPVCs useful in the present method are described in
the literature, as noted hereinabove, and are characterized more
particularly as progenitor cells extractable from the perivascular
region of umbilical cord, including but not limited to human
umbilical cord. Using the protocol described herein, it will be
appreciated that umbilical cord perivascular cells can also be
extracted from the umbilical cord vasculature of other mammals,
including horses, cows, pigs, primates and the like. The
perivascular region comprises the Wharton's jelly associated with
and external of the umbilical cord vasculature. The HUCPVCs are
extractable from the Wharton's jelly that lies in the perivascular
region, using standard methods of digestion such as with
collagenase or related enzymes suitable for removing associated
connective tissue, as described for instance by Sarugaser et al.,
2005, the entire contents of which are incorporated herein by
reference. Preferably, HUCPVCs are harvested only from the
perivascular cells, and not from Wharton's jelly extending beyond
the perivascular region, or from tissues or fluids that are part of
or internal to the vasculature itself. This avoids contamination by
other cells within the cord generally. In the alternative,
extraction from the Wharton's jelly without selection for
perivascular cells can be performed, provided the resulting cell
population is enriched for HUCPVCs using for instance flow
cytometry to enrich for progenitor cells having the phenotype and
characteristics noted herein. The HUCPVCs further are characterized
by relatively rapid proliferation, exhibiting a doubling time, in
each of passages 2-7, of about 20 hours (serum dependent) when
cultured under standard adherent conditions. Phenotypically, the
HUCPVCs are characterized, at harvest, as Oct 4-, CD14-, CD19-,
CD34-, CD44+, CD45-, CD649e+, CD90+, CD105(SH2)+, CD73(SH3)+,
CD79b-, HLA-G-, CXCR4+, c-kit+. In addition HUCPVCs contain cells
which are positive for CK8, CK18, CK19, PD-L2, CD146 and 3G5 (a
pericyte marker), at levels higher than in cell populations
extracted from Wharton's jelly sources other than the perivascular
region.
[0035] HUCPVCs can also express variable levels of MHC class I,
from 0-100% dependent upon manipulation. By subjecting harvested
cells to a freeze-thaw cycle, as described for instance by
Sarugaser et al., 2005, incorporated herein by reference, one
obtains a HUCPVC population that is substantially negative for both
MHC class I and MHC class II (95%). As used herein, the HUCPVCs are
considered to be "substantially" negative for MHC class I and MHC
class II if the number of cells resident in a given population and
expressing either one or both phenotypes is not more than about 20%
of the cell population, e.g., not more than 15%, 10%, 5% or less of
the total HUCPVC population. A determination can be made using
standard techniques of flow cytometry with appropriate tagged
antibody. This MHC double negative HUCPVC population is
particularly useful in the methods of the present invention. It
will be appreciated that, in the present method, the administered
HUCPVC population can comprise either freshly extracted (optionally
expanded) MHC class I negative cells, or the thawed MHC double
negative HUCPVCs. The MHC double negative HUCPVCs are far less
likely to stimulate an immune response in a recipient, and their
clinical use is accordingly preferred herein. It should be
appreciated, however, that manipulation of the MHC phenotype is not
essential. The immunoprivilege and immunomodulation properties are
seen also in freshly extracted HUCPVCs that have optionally been
stored, and not only in HUCPVCs that have been manipulated by
freeze/thaw.
[0036] In the present method, HUCPVCs are exploited for their
immunoprivileged and immunomodulatory properties, in clinical
setting that would benefit therefrom. The term "immunoprivileged"
is used herein with reference to cells, such as HUCPVCs, that when
incubated with peripheral blood lymphocytes, either do not
stimulate PBL proliferation to a statistically significant extent,
or retain their viability at a statistically significant level,
particularly when tested using the so-called one-way MLC assay
established in this art and exemplified herein.
[0037] The term "immunomodulatory" is used herein with reference to
the ability of HUCPVCs to mitigate, reduce or inhibit the reaction
between mismatched populations of lymphocytes, as revealed either
by a reduction in the mortality of a lymphocyte population, or by
an increase in the viability of a lymphocyte population, as
determined using, for instance, the so-called one- or two-way mixed
lymphocyte reaction (MLR).
[0038] It has in addition been found that factors exuded by
cultured HUCPVCs can alone exert an immunomodulatory effect, of the
type seen when intact HUCPVCs are used. Thus, in aspects and
embodiments of the present invention, the extracted soluble factors
produced upon culturing of HUCPVCs are used either alone or in
combination with the HUCPV cells, as immunomodulators.
[0039] The one or more immunomodulatory factors exuded upon HUCPVC
culturing are referred to herein a soluble factors, and are
extractable from the medium in which HUCPVCs are cultured. In one
embodiment, the immunomodulating soluble factors are provided as an
extract obtained when HUCPVC cells are removed from the medium
conditioned by their growth, such as by centrifugation. When
centrifugation is employed, the extract is provided as the
supernatant. Suitable HUCPVC culturing conditions are exemplified
herein. The extract is obtained by separating the cells from the
conditioned culturing medium, such as by centrifugation. In other
embodiments, the soluble factors are provided as an
immunomodulating fraction of such extract. An extract fraction
having immunomodulating activity is also useful herein, and can be
identified using the mixed lymphocyte reactions described herein.
These extract fractions can of course be obtained by fractionating
the HUCPVC extract using any convenient technique including solvent
extraction, HPLC fractionation, centrifugation, size exclusion,
salt or osmotic gradient fractionation and the like. Eluted or
collected fractions can then each be subjected to the MLR and
fractions active for immunomodulation can be identified, and a
fraction with immunomodulating activity can be used clinically in
the manner described herein.
[0040] Thus, in embodiments, the present invention comprises the
use of immunomodulating extracts or immunomodulating fractions
thereof comprising soluble factors exuded during culturing of
HUCPVCs.
[0041] Use of the HUCPVCs, and populations thereof that are
immunoprivileged and/or immunomodulatory, in accordance with the
present invention, entails their collection, optionally their
expansion, further optionally their cryogenic storage and revival
from the frozen state, and their subsequent formulation for
administration to the intended recipient. The particular
manipulation, dosing and treatment regimen will of course depend on
numerous factors, including the type and severity of the disease or
condition to be treated. For immunological conditions (eg. GvHD,
autoimmune conditions), the size of the HUCPVC population, i.e.,
the dose administered to the recipient, will lie generally in the
range from 0.01 to about 5 million cells per kilogram of recipient
body weight. For delivery, the cells are provided suitably as a
formulation further comprising a physiologically tolerable vehicle,
i.e., a vehicle that is tolerable not only by the cells but also by
the recipient. Suitably, the cells are provided in a sterile
formulation comprising, as carrier, a physiologically tolerable
vehicle such as saline, buffered saline such as PBS, cell culture
medium or similar liquid containing any of: essential amino acids,
growth factors, cytokines, vitamins, antibiotics or serum-free
chemically defined media etc, or sterile water. The formulated
cells can be administered by infusion, or by injection using for
instance volumes in the 1-25 mL range.
[0042] The immunomodulating soluble factors produced by HUCPVC and
extractable from spent HUCPVC culturing medium are similarly useful
in the manner described above with reference to intact HUCPVCs. In
one embodiment, the extract itself constitutes the pharmaceutical
composition, thus comprising the active agent in the form of
immunomodulating soluble factor, and the medium constituting the
physiologically tolerable vehicle. In the alternative, the extract
can be dried, to retain the soluble factor(s) and reconstituted in
a different vehicle, such as phosphate buffered saline. The dosage
size and dosing regimen suitable for clinical applications can be
determined with reference to the dosing effective for intact
HUCPVCs. The dose equivalent of extract can be determined by
calculating the relative potencies of the extract and intact cells
in the MLR assay, or any alternative thereto which is reflective of
the clinical environment in which the therapy will be exploited,
such as in appropriate animal models of the targeted
indication.
[0043] In use, the formulated HUCPVCs or soluble factors produced
thereby are administered to treat subjects experiencing or at risk
of developing an adverse immune reaction. Such subjects include
particularly subjects receiving or about to receive an allogeneic
or xenogenic transplant or graft, in the form of cells such as
marrow and peripheral blood, tissues including skin and vascular
tissue including coronary tissue and gastrointestinal tissue, or an
organ such as liver, kidney, heart, lung, etc. The formulated
HUCPVCs are useful particularly to reduce the onset or severity of
graft versus host disease, a condition resulting from an immune
attack of host tissues mediated by lymphocytes present in the donor
graft. In one embodiment of the invention, the HUCPVCs can be used
to treat the graft by incubation therewith for a period of time,
prior to transplantation, sufficient to reduce or arrest the
activity of lymphocytes resident in the graft. This incubation
would require HUCPVCs (from either fresh or frozen stock) to be
included at a dose of 5-60% of total graft lymphocytes (as
determined by the volume of blood present in the graft), preferably
10-40% for between 4 and 10 days, in order to halt proliferation
prior to implantation. In the case of an organ graft, the organ
would be incubated with HUCPVCs (suspended in a physiologically
tolerable vehicle as mentioned above), from either fresh or frozen
stock, at a dose of 0.01 to 5.times.10.sup.3 cells per gram mass of
the organ, prior to implantation, for a period of time to cause the
organ's lymphocytes to become inactive. For subjects that are graft
recipients, the HUCPVCs desirably are administered by in the tissue
directly surrounding the allogeneic organ to the recipient prior to
(e.g. within hours of), concurrently with, or after grafting (e.g.,
within hours after, and thereafter as necessary to control immune
reaction). The HUCPVCs can be administered, most suitably at the
site of the graft, such as by infusion or injection, either
subcutaneously, intramuscularly, intravasculary, intravenously,
intraarterially, or intraperitoneally. In one embodiment, the
recipient is treated at the time of grafting by infusion with
HUCPVC doses in the range from 5.times.10.sup.4 to 5.times.10.sup.7
cells per kg body weight, such as about 1 to 5.times.10.sup.6
cells. The cells are formulated in 10 ml of normal saline with 5%
human serum albumin. Two or more infusions can be used, each
lasting about 10-15 minutes. The cells can also be implanted in a
slow release formulation that allows the release of viable cells
over time at the implantation site (such as intraperitoneal,
intramuscular etc). Carriers suitable for this purpose include
gelatin, hyaluronic acid, alginate and the like. In another
embodiment of the invention, HUCPVCs can be utilized to treat
immunological conditions such as GvHD which are underway, and
possibly refractory to other treatments. The HUCPVCs or the exuded
soluble factors thus will be useful to treat subjects afflicted
with leukemias, aplastic anemias and enzyme or immune deficiencies
for whom the transplantation of immune cells or tissues containing
them are indicated.
[0044] The administration of HUCPVCs or the soluble factors also
has application in treating autoimmune diseases such as Crohn's
disease, lupus and multiple sclerosis, as well as rheumatoid
arthritis, type-1 insulin-dependent diabetes mellitus, adult
respiratory distress syndrome, inflammatory bowel disease,
dermatitis, meningitis, thrombotic thrombocytopernic purpura,
Sjogren's syndrome, encephalitis, uveitis, leukocyte adhesion
deficiency, rheumatic fever, Reiter' s syndrome, psoriatic
arthritis, progressive systemic sclerosis, primary biliary
cirrhosis, myasthenia gravis, lupus erythematosus, vasculitis,
pernicious anemia, antigen-antibody complex mediated diseases,
Reynard's syndrome, glomerulonephritis, chronic active hepatitis,
celiac disease, autoimmune complications of AIDS, ankylosing
spondylitis and Addison's disease. The administration of HUCPVCs in
this case is intravenously in a physiologically tolerable vehicle
(as mentioned previously), with a dose ranging from
0.1-10.times.10.sup.6 cells/kg body weight. More than one dose may
be required, and dosing can be repeated as needed.
[0045] Secondly, HUCPVCs can be used to treat protein/enzyme
deficiencies, wherein the HUCPVCs have been transfected with the
gene necessary to produce the desired protein/enzyme. This process
can include transduction (including but not limited to: lentiviral,
retroviral and adenoviral); and transfection (including but not
limited to: nucleofection, electroportation, liposomal) and are
transfused into a patient suffering from a deficiency. The dose
administered to the recipient will lie generally in the range from
0.01 to about 5 million cells per kilogram of recipient body
weight. HUCPVCs will then produce the protein or enzyme of interest
constitutively.
[0046] Finally, HUCPVCs can be engineered to introduce transgenes,
via the transfection methods mentioned above, for use as vaccines
in order to generate large quantities for administration to people
at risk of exposure to specific viruses/antigens.
MATERIALS AND METHODS
[0047] Cell Harvests
[0048] HUCPVCs
[0049] Ethical consent for this research was obtained from the
University of Toronto as well as Sunnybrook & Women's College
Health Sciences Centre. Umbilical cords were collected from aseptic
caesarean births of full term babies, upon obtaining informed
consent from both parents. The cords were immediately transported
to the University of Toronto where cells were extracted from the
perivascular area under sterile conditions as reported
previously.sup.16. Briefly, 4 cm sections of cord were cut, and the
epithelium was removed. The vessels were then extracted including
their surrounding Wharton's jelly, tied in a loop to prevent smooth
muscle digestion, and digested overnight in a collagenase solution.
Upon removal from the digest the following day, the cells were
rinsed in ammonium chloride to lyse any red blood cells from the
cord blood. Following this, the cells were rinsed and plated out in
85% .alpha.-MEM containing 5% fetal bovine serum and 10%
antibiotics (penicillin G at 167 units/ml; Sigma, gentamicin 50
.mu.g/ml; Sigma, and amphotericin B 0.3 .mu.g/ml) at a density of
4,000 cells/cm.sup.2. The cells are passaged when they reach 75-80%
confluence, which is approximately every 6-7 days.
[0050] MHC -/- HUCPVCs
[0051] Test cell populations of >1.times.10.sup.5 cells were
washed in PBS containing 2% FBS and re-suspended in PBS+2% FBS with
saturating concentrations (1:100 dilution) of the following
conjugated mouse IgG1 l HLA-A,B,C-PE (BD Biosciences #555553, Lot
M076246) (MHC I), HLA-DR,DP,DQ-FITC (BD Biosciences #555558, Lot
M074842) (MHC II) and CD45-Cy-Cychrome (BD Biosciences #555484, Lot
0000035746) for 30 minutes at 4.degree. C. The cell suspension was
washed twice with PBS+2% FBS and re-suspended in PBS+2% FBS for
analysis on a flow cytometer (XL, Beckman-Coulter, Miami, Fla.)
using the ExpoADCXL4 software (Beckman-Coulter). Positive staining
was defined as the emission of a fluorescence signal that exceeded
levels obtained by >99% of cells from the control population
stained with matched isotype antibodies (FITC-, PE-, and
Cy-cychrome-conjugated mouse IgG1,.kappa. monoclonal isotype
standards, BD Biosciences), which was confirmed by positive
fluorescence of human BM samples. For each sample, at least 10,000
list mode events were collected. All plots were generated in EXPO
32 ADC Analysis software.
[0052] The attached cells were sub-cultured (passaged) using 0.1%
trypsin solution after 7 days, at which point they exhibited 80-90%
confluency as observed by light microscopy. Upon passage, the cells
were observed by flow cytometry for expression of MHC-A,B,C,
MHC-DR,DP,DQ, and CD45. They were then plated in T-75 tissue
culture polystyrene flasks at 4.times.10.sup.3 cells/cm.sup.2 in
SM, and treated with 10.sup.-8M Dex, 5 mM .beta.-GP and 50 .mu.g/ml
ascorbic acid to test the osteogenic capacity of these cells. These
flasks were observed on days 2, 3, 4, 5 and 6 of culture for CFU-0,
or bone nodule, formation. Any residual cells from the passaging
procedure also were cryopreserved for future use.
[0053] Aliquots of 1.times.10.sup.6 PVT cells were prepared in 1 ml
total volume consisting of 90% FBS, 10% dimethyl sulphoxide (DMSO)
(Sigma D-2650, Lot#11K2320), and pipetted into 1 ml polypropylene
cryo-vials. The vials were placed into a -70.degree. C. freezer
overnight, and transferred the following day to a -150.degree. C.
freezer for long-term storage. After one week of cryo-preservation,
the PVT cells were thawed and observed by flow cytometry for
expression of MHC-A,B,C, MHC-DR,DP,DQ, and CD45. A second protocol
was used in which the PVT cells were thawed after one week of
cryopreservation, recultured for one week, sub-cultured then
reanalyzed by flow cytometry for expression of MHC-A,B,C,
MHC-DR,DP,DQ, and CD45.
[0054] It was noted that the frequency of MHC -/- within the fresh
cell population is maintained through several passages. When fresh
cells are frozen after passaging, at -150.degree. C. for one week
and then immediately analyzed for MHC phenotype, this analyzed
population displays a remarkably enhanced frequency of cells of the
MHC -/- phenotype. In particular, first passage of cryopreserved
cells increases the relative population of MHC -/- cells to greater
than 50% and subsequent freezing and passaging of those cells
yields an MHC -/- population of greater than 80%, 85%, 90% and
95%.
[0055] Lymphocytes
[0056] Peripheral blood lymphocytes (PBLs) were extracted from
heparinized blood from healthy donors. Cell separation was achieved
by Ficoll-Paque.TM. PLUS density gradient (Amersham Biosciences
#17-1440-03) in which the cells were spun for 35 minutes at
380.times.g. The buffy coat was removed and counted using a
ViCell-XR.TM. (Beckman Coulter) with a protocol specific for
lymphocytes as determined by cell and nucleus size. The cells were
then plated out as per the requirements of the assay in 80%
RPMI-1640 media (Sigma #R5886) containing HEPES (25 mmol/L),
L-Glutamine (2 mmol/L), 10% fetal bovine serum and 10%
antibiotics.
[0057] Mixed Lymphocyte Cultures
[0058] Mitomycin C
[0059] In order for one-way MLCs to be performed, the HUCPVCs and
one of the PBL populations have to remain quiescent. This is
achieved by treating the cells with mitomycin C (MMC) at a set
concentration and time, allowing the MMC to adhere to the DNA and
prevent division. This concentration was determined by a titration
curve of MMC incubated for 20 minutes at 37.degree. C. (5%
CO.sub.2) with a starting cell population of 5000 cells in a well
of a 96 well plate. Concentrations of 10, 20, 30, 40, 50, and 75
.mu.g/mL of MMC were tested in regular media (85% .alpha.-MEM, 5%
FBS, 10% antibiotics) and washed twice with PBS afterwards to
remove any traces of the MMC. The wells were counted after a week
to assess proliferation. It is essential that all traces of MMC are
removed so it does not affect the proliferative capacity of
lymphocytes when the cell populations are combined in an MLC. To
ensure this, empty wells of a 96 well plate (Falcon) were treated
with MMC and washed as per the protocol. Lymphocytes were then
added and assayed for their proliferation compared to control
normal wells.
[0060] Immunoprivilege Assays
[0061] Triplicates of 1.times.10.sup.4 HUCPVCs (both fresh and
frozen have been assayed) were plated in 96 well plates (n=5). Once
the cells had attached (after approximately 2 hours), they were
treated with MMC at 20 .mu.g/mL. The HUCPVCs were then rinsed, and
10.sup.5 PBLs from Donor 1 were added to each well. The plates were
incubated at 37.degree. C. with 5% CO.sub.2 air in 80% RPMI-1640
media containing HEPES (25 mmol/L), L-Glutamine (2 mmol/L), 10%
fetal bovine serum and 10% antibiotics. After 6 days the
lymphocytes present in culture with HUCPVCs were counted using the
ViCell counter, and compared to controls.For the cell death assay,
plates were allowed to incubate for four hours, and HUCPVCs were
assessed for early and late stage cell death markers; annexin 5
(R&D Systems TA4638) and 7-amino-actinomycin D (7-AAD)
respectively. These levels were measured and compared using Flow
Cytometry on a Beckman Coulter FlowCenter. For the PBL
proliferation assay, the cells were allowed to incubate for 6 days,
after which they were stained with 5-bromo-2-deoxyuridine (BrdU), a
base analog of thymidine, and measured using flow cytometry.
Controls of PBLs alone and HUCPVCs alone were used for both
assays.
[0062] For one-way MLCs, HUCPVCs were first plated in a 96 well
plate in triplicate (1, 2, 3, and 4.times.10.sup.4 cells per well),
treated with MMC and washed with PBS. PBLs were retrieved from two
unmatched donors selected from a pool of potential donors (Mismatch
on 5 out of 6 HLA tested: Donor 1 HLA-A *01, *02; B *07, *18; DRB1
*15, *--; Donor 2 A *01, *--; B *08, *--; DRB1 *03, *--). Typing
was performed at the Regional Histocompatibility Laboratory
(Toronto General Hospital, Toronto, ON) using DNA assignment
techniques at low resolution. After Ficolling, Donor 2's
lymphocytes were treated with MMC to be quiescent. The cells were
then spun down and washed, and added to a 96 well plate at 10.sup.5
cells per well. Donor 1's lymphocytes were added at 10.sup.5 cells
per well and the three cell populations were allowed to co-incubate
for 6 days, after which they were stained with
5-bromo-2-deoxyuridine (BrdU), a base analog of thymidine. Flow
cytometry for BrdU was then performed on the lymphocytes to assay
proliferation. Following this, a similar assay was performed with
the result being measured using daily counts of lymphocytes (from
day 1 to 6) using the ViCell-XR.TM. cell counter, with a
lymphocyte-specific protocol. All results were compared to
allogeneic and autologous controls from both donors.
[0063] Immunomodulation Assays
[0064] Two-way MLCs incorporate two PBL populations from unmatched
donors (same donors as above), both of which are permitted to
proliferate. Briefly, 1.times.10.sup.5 PBLs from both donors were
added to each well of a 96 well plate and left to incubate for six
days. In the course of the experiment, either 1 or 4.times.10.sup.4
HUCPVCs were added to wells in triplicate on days 0, three or five
in order to analyze the effectiveness of HUCPVCs if an immune
reaction has already begun. The 10 and 40% HUCPVC:PBL ratios were
chosen as both had showed positive results previously, and thus
were chosen as the low and high levels of HUCPVC inclusion. Samples
from each plate were counted every day using the ViCell-XR.TM. cell
counter and compared to autologous and allogeneic controls.
[0065] Soluble Factor
[0066] The two-way MLC assay was performed again, without direct
HUCPVC to PBL cell contact to determine if the effect noted was due
to a soluble factor, or if cell-cell contact was necessary. The
HUCPVCs (1 and 4.times.10.sup.4 cells per well) were cultured on a
Transwell.RTM. insert (Corning) for a 24 well plate, and allowed to
attach for approximately 2 hours. Once they had attached, the
insert was transferred to the 24 well plate that contained a
co-culture of PBL populations from Donor 1/Donor 2 (same donors as
above) (n=3). The lymphocyte cell numbers were counted daily for
six days and compared to autologous and allogeneic controls using
the ViCell-XR.TM..
[0067] Lymphocyte Activation
[0068] Both immunoprivilege assays and two-way MLCs were performed
as mentioned previously. The endpoint of this assay was flow
cytometric analysis of the lymphocytes for the presence of IL-2R
(CD25) (Becton Dickinson, #555431), a marker of activated
lymphocytes. This assay was performed over 6 days to determine if
HUCPVCs caused an increase or decrease in lymphocyte activation.
Lymphocytes were also co-stained with CD45 to ensure proper cell
population was obtained. Negative controls were lymphocyte cultures
with no HUCPVCs added, and unstained.
[0069] Activated T Cell Line Generation
[0070] PBLs were extracted from Donor 1 and 2 as previously, and
separated using the Ficoll gradient. The cells were counted, and
cells from Donor 2 were rendered quiescent with MMC. 10.sup.6 Cells
from Donor 1 were plated in a 24 well plate, and stimulated with a
1:1 ratio of quiescent PBLs from Donor 2. The cells were fed with 2
mLs of RPMI-1640 media (supplemented with 10% serum and 10%
supplements as previously) and allowed to activate. Media was
changed upon a perceptible change of its colour to yellow (.about.3
days). After .about.11 days (or when the media changed colour in
under 3 days), the cells were harvested and counted. They were
re-plated at 10.sup.6 per well, and re-stimulated with a 1:1 ratio
of quiescent PBLs from Donor 2. Upon the second stimulation, IL-2
was added at a concentration of 100 U/mL (BD Biosciences #354043),
every 2 days with feeding. When media turned yellow before 3 days,
the cells were harvested, counted and split. Following this
procedure, the cells were ready to be used as Activated T
lymphocytes (ATLs), with specific antibodies to Donor 2. The PBL
co-culture and two-way MLCs were carried out as previously and
assayed for cell proliferation and expression of CD25.
[0071] Lymphocyte Labelling
[0072] In order to visualize, and quantify, the difference between
two lymphocyte populations, PKH26 was used (Sigma #PKH26-GL). PHK26
is a non-cytotoxic membrane dye with a long half life (-100 days).
Cells were stained as per the protocol supplied with the product:
lymphocytes were trypsinized, pooled, and pelleted; the diluted dye
was then added to the cell suspension for 2 to 5 minutes (2 mL of
2.times.10.sup.-6 molar PHK26 solution). After staining, an equal
volume of serum was added to halt the reaction; the cells were
suspended in media, spun down and washed several times. The stain
was then visualized on the fluorescence microscope to ensure
appropriate dye uptake resulted. The cells used for staining were
the ATLs obtained from Donor 1; these red cells were included in an
MLC with unstained cells from Donor 2, and HUCPVCs. The endpoint of
the assay was flow cytometry for CD45 (BD #555482) and CD25, gated
on the presence or absence of PKH26. Negative controls were
unstained cells, and MLCs with no HUCPVCs
[0073] Transfection
[0074] First, 293 Cells are transfected with the desired DNA and
plasmids (vector DNA, 10 .mu.g gag/pol expressing plasmid, 10 .mu.g
of rev expressing plasmid, 10 .mu.g of tat expressing plasmid, 5
.mu.g of VSV-G expressing plasmid with 2.5 M CaCl.sub.2). These are
allowed to incubate overnight, after which the media is changed.
Cells are then left with this media for three more days, after
which the supernatant of the cells is collected and filtered. The
viral supernatant is then concentrated with ultracentrifugation
(50000 g for 90 minutes) or using an Amicon Ultra-15 Centrifugal
Filter device (100,000 MWCO; Millipore). When this process is
complete, the viral supernatant can be combined with the HUCPVCs at
a concentration determined by titering the concentrated virus, and
allowed to incubate overnight. The following day, more media is
added, and the cells incubate for 6 more hours before changing the
media. In this manner, HUCPVCs can be engineered to introduce and
express a transgene that encodes any protein, including proteins
useful to manage adverse immune reactions (immunosuppressive
proteins). Such proteins include CTLA4, VCP, PLIF, LSF-1, Nip,
CD200 and Uromodulin.
[0075] Microarray Analysis
[0076] The Oligo GEArray Human Cancer Microarray (Superarray
Biosciences, Frederick Md., Cat#: OHS-802) was used to find changes
in the expression of genes representative of several different
pathways frequently altered during the progression of cancer The
Oligo GEArray for human cancer has 440 representative cancer genes
and is organized into functional gene groupings including
apoptosis, cell cycle, cell growth and differentiation, signal
transduction, and other cancer-related genes.
[0077] HUCPVCs and human bone marrow-derived MSCs were grown to
passage 2 and RNA was isolated from these cells. Purified RNA was
processed according to manufacturer's protocol (Superarray
Biosciences Corp.) and hybridized to Oligo GEArray Human Cancer
microarrays. The Oligo GEArray Human Cancer Microarray was used to
determine the differential expression of genes related to cancer in
HUCPVCs compared with normal human bone marrow-derived MSCs.
RESULTS
[0078] Mitomycin C is an Effective Anti-Proliferative Agent on
HUCPVCs
[0079] HUCPVCs were treated with a range of concentrations of MMC
for 20 minutes at 37.degree. C. (5% CO.sub.2). FIG. 1 shows the
cell numbers of HUCPVCs after one week in culture post-MMC
treatment (n=2). All cells show a marked decrease in proliferation
relative to control (p <0.001), with no difference among
treatments. Thus, 20 .mu.g/mL was chosen in accordance with the
literature. FIG. 2 illustrates the lack of effect of cells plated
in wells treated with MMC and washed, versus untreated wells (n=2,
p=0.16). Therefore, the MMC will not have an effect on experimental
results obtained in wells previously treated with MMC. All
statistics presented herein were obtained using ANOVAs to compare
means via the R Project for Statistical Computing.
[0080] HUCPVCs are not Recognized as Foreign by Lymphocytes
[0081] Upon co-incubation of a HUCPVC population with lymphocytes
(Donor 1), there was a statistically significant increase in HUCPVC
death at a proportion of 10% HUCPVC:PBL (n=5, p=0.01), as measured
by annexin 5 mean fluorescence intensity (MFI). FIG. 3 shows this
increased cell death was not noted at HUCPVC doses higher than 10%,
thus at the correct proportion, HUCPVCs are not attacked by
unmatched lymphocytes. This was confirmed using a lymphocyte
proliferation assay in which it can be seen that lymphocytes do
proliferate in response to 10% HUCPVC:PBL, as measured by BrdU MFI
(p=0.02), but not at higher HUCPVC concentrations (FIG. 4).
Lymphocyte proliferation is a standard measure of activation, as
division of both T and B lymphocytes occurs in the activation
cascade. Therefore at a lower proportion, HUCPVCs do not provide
enough of a presence such that their immunological avoidance
capabilities are realized. However at higher concentrations,
20-40%, the PBLs do not proliferate, and the HUCPVCs are not
killed.
[0082] HUCPVCs were also analyzed for their effect on lymphocyte
cell number upon inclusion in a co-culture with resting PBLs or
ATLs. In both cases, HUCPVCs caused no significant increase over
control cell number (PBL: 35.2.+-.3.1.times.10.sup.3, +10% HUCPVCs
45.0.+-.5.7.times.10.sup.3; ATL: 38.8.+-.18.2.times.10.sup.3, +10%
HUCPVCs 40.8.+-.4.8.times.10.sup.3), indicating their
immunoprivilege in either resting or stimulated conditions (FIG.
7).
[0083] HUCPVCs were included in a one-way MLC in proportions of 10,
20, 30 and 40% of the PBL population and assessed for their
proliferation by BrdU expression after 6 days. FIG. 8 shows no
significant increase in the number of cells proliferating
regardless of the proportion of HUCPVCs included.
[0084] HUCPVCs are Immunomodulatory
[0085] FIG. 5 shows that on day 6 the allogeneic co-cultured
lymphocytes have increased in number, whereas all cultures with
HUCPVCs present; regardless of when they were added or the
proportion added, have a significantly lower lymphocyte cell count
than the control (n=3).
[0086] HUCPVCs Can Exert their Action through a Soluble Factor
[0087] TransWell.degree.0 inserts were used to separate HUCPVCs
from PBLs in a two-way MLC. No significant reduction in lymphocyte
number relative to control was seen within any day with either 10%
or 40% HUCPVCs (n=3) (FIG. 6). However, upon increasing the sample
number, the addition of 10% HUCPVCs showed a significant reduction
in lymphocyte cell number over a 6 day culture period compared to
control (MLC: 40.7.+-.32.9.times.10.sup.3 cells, 10% HUCPVCs: 21.3
+14.7.times.10.sup.3 cells) (FIG. 9). Soluble factor(s) may
therefore contribute to HUCPVC immunomodulation, however what that
factor(s) is/are and how they affect lymphocytes is still
unknown.
[0088] HUCPVCs Reduce the Activation State of Lymphocytes
[0089] ATLs stained with PKH26 were added in a co-culture with 10%
HUCPVCs, and assayed for their expression of CD25 (IL-2 receptor),
a marker of lymphocyte activation. Upon inclusion of HUCPVCs, both
the percentage of cells expressing CD25 (Control: 100%.+-.0, 10%
HUCPVC: 96.9%.+-.0.7), and the mean fluorescence intensity
(Control: 28.6.+-.0.1, 10% HUCPVC: 3.76.+-.0.1) was significantly
reduced (FIG. 10). Thus, HUCPVCs have a physical effect on
activated lymphocytes, by reducing their activation state.
[0090] In addition, HUCPVCs reduced the expression of CD45 of the
lymphocytes, both the percentage (Control: 100%+0, 10% HUCPVC:
99.6%+0.1, 40% HUCPVC: 98.4%+0.36) and the mean fluorescence
intensity (Control: 28.20+4.24, 10% HUCPVC: 16.33+1.27, 40% HUCPVC:
14.70.+-.1.22) were significantly different (p<0.05) (FIG. 11).
These results were unexpected as CD45 is expressed on all
lymphocytes. However it has been seen that CD45 is crucial for the
development and function of lymphocytes.sup.36, and may be a
further indication of the reduced function of the lymphocytes due
to the addition of the HUCPVCs.
[0091] Transfection
[0092] HUCPVCs were successfully transfected with GFP, and the
cells expressed high levels of the protein. A transfection
efficiency of 97% was achieved (FIG. 12), with maintenance of good
proliferative rates. This success rate varies according to the
passage at which the cells are transfected. With a functioning
transfection protocol, it is thus possible to transfect the cells
with any protein and have it constitutively expressed.
[0093] Microarray Analysis
[0094] HUCPVCs do not express any detectable levels of genes
associated with tumorigenesis. The gene array analysis resulted in
the absence of functional genes associated with human cancer and
expressed genes known to be important, for example, in cell cycle
regulation, such as Cyclin D1 (CCND1), CCND2 and CCND3 (FIG.
13).
DISCUSSION
[0095] Herein described are the in vitro immunoprivileged and
immunomodulatory properties of an MSC population from a source
other than bone marrow, the human umbilical cord. HUCPVCs are
extracted from the perivascular area of the cord, as this was
believed to be the most rapidly proliferating population of cells.
Previously, it was shown that endothelial cells from the wall of
the umbilical cord vein stimulated lymphocytes in vitro.sup.37 .
This is in stark contrast to the results reported, and reinforces
the distinct area from which HUCPVCs are retrieved.
[0096] HUCPVCs thus are well suited for clinical use particularly,
but not only, to reduce the onset and/or severity of graft versus
host disease, and to reduce or eliminate graft rejection by the
host, and for the treatment of other immune-mediated disorders that
would benefit from suppression of a mixed lymphocyte reaction. In
addition, when manipulated by transfection to contain the gene of a
protein/enzyme of interest, HUCPVCs are able to produce this
product constitutively, and would thus be useful in the treatment
of any condition in which a protein/enzyme deficiency results in a
detrimental effect to the patient, especially as they can be used
allogeneically in a mismatched patient without rejection. In
addition, HUCPVCs can also be used to generate vaccines of interest
after transfection with the necessary gene.
[0097] As progenitor cells having the propensity to expand and
differentiate over time into various mesenchymal tissues dictated
by their growth environment, HUCPVCs like other mesenchymal
progenitor or stem cells may carry some risk that their growth and
differentiation in vivo will not be controlled. Remarkably, as a
further benefit of the use of HUCPVCs clinically, it has been
determined that the HUCPVCs exhibit extremely low telomerase
activity, an indicator of their propensity for tumorigenesis.
Furthermore, it has been determined that HUCPVCs lack many of the
genetic markers that are hallmarks of tumorigenesis. Tumorigenesis
occurs by mutations that deregulate biological pathways and cause
cells to grow and divide unchecked, to avoid apoptosis (programmed
cell death), to respond abnormally to growth factors, to receive
blood supply (angiogenesis), and to migrate from one location to
another (metastasis and invasiveness). Many genes are involved in
each of these control mechanisms, and a mutation in any one of them
can cause deregulation.
[0098] The following references are incorporated hereby by
reference in their entirety:
REFERENCE LIST
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References