ISOLATION OF PRECURSOR CELLS FROM HEMATOPOIETIC AND NON-HEMATOPOIETIC TISSUES ANDTHEIR USE
BACKGROUND OF THE INVENTION
5 The present invention generally relates to the
isolation of precursor cells and their use in bone and
cartilage regeneration procedures and, more
particularly, is concerned with a direct method for
isolating precursor cells from a variety of body tissue
10 types utilizing cell surface antigen CD34 and other
precursor cell surface antigens on CD34+ cells.
Osteogenesis and chondrogenesis are highly complex
biological processes having considerable medical and
clinical relevance. For example, more than 1,400,000
15 bone grafting procedures are performed in the developed
world annually. Most of these procedures are
administered following joint replacement surgeries, or
during trauma surgical reconstructions. The success or
failure of bone grafting procedures depends largely on
20 the vitality of the site of grafting, graft processing,
and in the case of allografts, on immunological
compatibility between donor and host. Compatibility
issues can largely be negated as an important
consideration in the case of autologous grafting
procedures, which involve taking bone tissue from one
site of the patient for transplantation at another
site. While autologous bone grafts are generally
successful they do require additional surgery in order
to harvest the graft material, and not uncommonly are
accompanied by post-operative pain, hemorrhage and
infection.
Cartilage regeneration and replacement procedures
are perhaps even more problematic. Unlike osteogenesis,
chondrogenesis does not typically occur to repair
damaged cartilage tissue. Attempts to repair damaged
cartilage in any clinically meaningful fashion have met
with only limited success. In many cases, the most
effective treatment for cartilage damage is prosthetic
joint replacement.
These and other difficulties w th presently
available bone-grafting and cartilage regeneration
procedures have prompted intensive investigations into
the cellular and molecular bases of osteogenesis and
chondrogenesis. Some promising research to date has
been in the identif cation and isolation of bone and
cartilage precursor cells from marrow and other
tissues.
Early investigations into the complexity of bone
marrow demonstrated that lethally irradiated animals
could be rescued by marrow transplants, suggesting that
bone marrow contained a restorative factor having the
capacity to regenerate the entire hematopoietic system.
More recent experiments have shown that marrow also has
the capacity to regenerate bone and other mesenchymal
tissue types when implanted in vivo in diffusion
chambers. (See e.g. A. Friedenstein etal. "Osteogenesis
in transplants of bone marrow cells." J. Embryol. Exp.
Morph. 16, 381-390, 1960; M. Owen. " The osteogenic
potential of marrow." UCLA Symp. on Mol. and Cell.
Biol. 46, 247-255, 1987) Results of this nature have
led to the conclusion that bone marrow contains one or
more populations of pluripotent cells, known as stem
cells, having the capacity to differentiate into a wide
variety of different cell types of the mesenchymal,
hematopoietic, and stromal lineages.
The process of biological differentiation, which
underlies the diversity of cell types exhibited by bone
marrow, is the general process by which specialized,
committed cell types arise from less specialized,
primitive cell types. Differentiation may conveniently
be thought of as a series of steps along a pathway, in
which each step is occupied by a particular cell type
potentially having unique genetic and phenotypic
characteristics. In the typical course of
differentiation a pluripotent stem cell proceeds
through one or more intermediate stage cellular
divisions, ending ultimately in the appearance of one
or more specialized cell types, such as T lymphocytes
and osteocytes. The uncommitted cell types which
precede the fully differentiated forms, and which may
or may not be true stem cells, are defined as precursor
cells.
Although the precise signals that trigger
differentiation down a particular path are not fully
understood, it is clear that a variety of chemotactic,
cellular, and other environmental signals come into
play. Within the mesenchymal lineage, for example,
mesenchymal stem cells (MSC) cultured in vitro can be
induced to differentiate into bone or cartilage in vivo
and m vitro, depending upon the tissue environment or
the culture medium into which the cells are placed.
(See e.g. S Wakitani et al. "Mesenchymal cell-based
repair of large, full-thickness defects of articular
cartilage" J. Bone and Joint Surg, 76-A, 579-592
(1994); J Goshima, VM Goldberg, and AI Caplan, "The
osteogenic potential of culture-expanded rat marrow
mesenchymal cells assayed in vivo in calcium phosphate
ceramic blocks" Clin. Orthop. 262, 298-311 (1991) ; H
Nakahara etal "In vitro differentiation of bone and
hypertrophic cartilage from periosteal-derived cells"
Exper. Cell Res. 195, 492-503 (1991)) .
Studies of this type have conclusively shown that
MSC are a population of cells having the capacity to
differentiate into a variety of different cell types
including cartilage, bone, tendon, ligament, and other
connective tissue types. Remarkably, all distinct
mesenchymal tissue types apparently derive from a
common progenitor stem cell, viz. MSC. The MSC itself
is intimately linked to a trilogy of distinctly
differentiating cell types, which include
hematopoietic, mesenchymal, and stromal cell lineages.
Hematopoietic stem cells (HSC) have the capacity for
self-regeneration and for generating all blood cell
lineages while stromal stem cells (SSC) have the
capacity for self-renewal and for producing the
hematopoietic microenvironment .
It is a tantalizing though controversial prospect
whether the complex subpopulations of cell types
present in marrow (i.e. hematopoietic, mesenchymal, and
stromal) are themselves progeny from a common ancestor.
The search for ancestral linkages has been challenging
for experimentalists. Identifying relatedness among
precursor and stem cell populations requires the
identification of common cell surface markers, termed
"differentiation antigens, " many of which appear in a
transitory and developmentally-related fashion during
the course of differentiation. One group, for example,
has reported an ancestral connection among MSC, HSC,
and SSC, though later issued a partial retraction (S.
Huang & L. Terstappen. "Formation of haematopoietic
microenvironment and haematopoietic stem cells from
single human bone marrow stem cells" Nature, 360, 745-
749, 1992; L. Terstappen & S. Huang. "Analysis of bone
marrow stem cell" Blood Cells, 20, 45-63, 1994; EK
Waller etal. "The common stem cell hypothesis
reevaluated: human fetal bone marrow contains separate
populations of hematopoietic and stromal progenitors"
Blood, 85, 2422-2435, 1995) . However, studies by
another group have demonstrated that murine osteoblasts
possess differentiation antigens of the Ly-6 family.
That finding is significant in the present context
because the Ly-6 antigens are also expressed by cells
of the murine hematopoietic lineage. (M.C. Horowitz
etal. "Expression and regulation of Ly-6
differentiation antigens by murine osteoblasts"
Endocrinology, 135, 1032-1043, 1994) Thus, there may
indeed be a close lineal relationship between
mesenchymal and hematopoietic cell types which has its
origin in a common progenitor. A final answer on this
question must await further study.
One of the most useful differentiation antigens
for following the course of differentiation in human
hematopoietic systems is the cell surface antigen known
as CD34. CD34 is expressed by about 1% to 5% of normal
human adult marrow cells in a developmentally, stage-
specific manner [CI Civin etal. "Antigenic analysis of
hematopoiesis. Ill . A hematopoietic progenitor cell
surface antigen defined by a monoclonal antibody raised
against KG-la cells. J Immunol, 133, 157-165, 1984] .
CD34+ cells are a mixture of immature blastic cells and
a small percentage of mature, lineage-committed cells
of the myeloid, erythroid and lymphoid series. Perhaps
1% of CD34+ cells are true HSC with the remaining
number being committed to a particular lineage. Results
n humans have demonstrated that CD34+ cells isolated
from peripheral blood or marrow can reconstitute the
entire hematopoietic system for a lifetime. Therefore,
CD34 is a marker for HSC and hematopoietic progenitor
cells .
While CD34 is widely recognized as a marker for
hematopoietic cell types, it has heretofore never been
recognized as a reliable marker for precursor cells
having osteogenic potential m vivo. On the contrary,
the prior art has taught that bone precursor cells are
not hematopoietic origin and that bone precursor
cells do not express the hematopoietic cell surface
antigen CD34 (MW Long, JL Williams, and KG Mann
"Expression of bone-related proteins in the human
hematopoietic microenvironment" J. Clm. Invest. 86,
1387-1395, 1990; MW Long etal. "Regulation of human
bone marrow-derived osteoprogenitor cells by osteogenic
growth factors" J. Clm. Invest. 95, 881-887, 1995; SE
Haynesworth et al. "Cell surface antigens on human
marrow-derived mesenchymal cells are detected by
monoclonal antibodies" Bone, 13, 69-80, 1992) .
To date, the most common sources of precursor
cells having osteogenic potential have been periosteum
and marrow. Many researchers use cells isolated from
periosteum for in vitro assays (See e.g. I Binderman et
al. "Formation of bone tissue in culture from isolated
bone cells" J. Cell Biol. 61, 427-439, 1974) . The
pioneer of the concept of culturing bone marrow to
isolate precursor cells for studying bone and cartilage
formation is A.J. Friedenstein. He developed a culture
method for isolating and expanding cells (CFU-f) from
bone marrow which can form bone (AJ Friedenstein et al.
"The development of fibroblast colonies in monolayer
cultures of guinea pig bone marrow and spleen cells"
Cell Tiss. Kinet. 3, 393-402, 1970) . Others have used
Friedenstein' s culture system extensively to study the
origin of osteoblasts (See e.g. M. Owen, "The origin of
bone cells in the postnatal organism" Arthr. Rheum. 23,
1073-1080, 1980) . Friedenstein showed that CFU-f cells
from marrow will form bone, cartilage, and fibrous
tissue when implanted, though CFU-f cells cultured from
other sources such as thymus, spleen, peripheral blood,
and peritoneal fluid will not form bone or cartilage
without an added inducing agent. Friedenstein recently
discussed the pos'sible clinical utility of CFU-f and
pointed out some obstacles that must be overcome, such
as the need for culturing for several passages and
developing a method for transplanting the cells (AJ
Friedenstein "Marrow stromal fibroblasts" Calcif. Tiss.
Int. 56(S) : S17, 1995) .
Similarly, the most common sources of cartilage
precursor cells to date have been periosteum,
perichondrium, and marrow. Cells isolated from marrow
have also been used to produce cartilage in vivo (S
Wakitani et al . "Mesenchymal cell-based repair of
large, full-thickness defects of articular cartilage"
J. Bone and Joint Surg, 76-A, 579-592 (1994) .
Periosteal and perichondral grafts have also been used
as sources of cartilage precursor cells for cartilage
repair (SW O'Driscoll et al. "Durability of regenerated
articular cartilage produced by free autogenous
periosteal grafts in major full-thickness defects in
joint surfaces under the influence of continuous
passive motion" J. Bone and Joint Surg. 70A, 1017-1035,
1986; R Coutts et al. "Rib perichondral autografts in
full-thickness articular defects in rabbits" Clin.
Orthop. Rel. Res. 275, 263-273, 1992) .
In a series of patents, Caplan etal. disclose a
method for isolating and amplifying mesenchymal stem
cells (MSC) from marrow. (U.S. Patents 4,609,551;
5,197,985; and 5,226,914) The Caplan method involves
two basic steps: 1) harvesting marrow and 2) amplifying
the MSC contained in the harvested marrow by a 2 to 3
week period of in vitro culturing. This method takes
advantage of the fact that a particular culture medium
favors the attachment and propagation of MSC over other
cell types. In a variation on this basic method, MSC
are first selected from bone marrow using specific
antibodies against MSC prior to in vitro culturing.
(Caplan and Haynesworth; WO 92/22584) The in vitro
amplified, marrow-isolated MSC may then be introduced
into a recipient at a transplantation repair site. (A.
Caplan. "precursor cells cells" J. Ortho. Res. 9, 641,
1991; S.E.Haynesworth, M.A.Baber, and A.I. Caplan.
"Cell surface antigens on human marrow-derived
mesenchymal cells are detected by monoclonal
antibodies," Bone, 13, 69-80, 1992)
The current methods used to isolate precursor
cells have a number of drawbacks to consider. First,
the methods require that bone marrow or other tissues
be harvested. Harvesting bone marrow requires an
additional surgical procedure with the appendant
possibility of complications from anesthesia,
hemorrhage, infection, and post-operative pain.
Harvesting periosteum or perichondrium is even more
invasive. Second, the Caplan method requires a
substantial period of time (2 to 3 weeks) for in vitro
culturing of marrow-harvested MSC before the cells can
be used in further applications. This additional cell
culturing step renders the method time-consuming,
costly, and subject to more chance for human error.
Consequently, a need exists for a quicker and
simpler method for identifying and isolating precursor
cells having osteogenic and chondrogenic potential
which can be used for in vivo bone and cartilage
regeneration procedures.
SUMMARY OF THE INVENTION
It is an object of this invention to provide a
method for isolating precursor cells having the
potential to generate bone or cartilage from a variety
of hematopoietic and non-hematopoietic tissues.
It is also an object of this invention to provide
a method for isolating precursor cells having the
potential to generate bone or cartilage from peripheral
blood, marrow, or adipose tissue based on binding by a
reagent to cell surface antigen CD34 or other surface
antigens on CD34+ cells.
It is another object of this invention to provide
a method for isolating precursor cells having the
potential to generate bone or cartilage from adipose
tissue based on sedimentation differences in the cells
comprising the tissue.
It is a further object of the present invention to
provide a method for in vivo bone and cartilage
regeneration involving transplantation with CD34+
precursor cells isolated from peripheral blood, marrow,
or adipose tissue.
It is a still further object of the present
invention to provide a direct, single-step method for
in vivo bone or cartilage regeneration involving the
isolation of CD34+ precursor cells from peripheral
blood, marrow, or adipose tissue and immediate
implantation at a connective tissue site needing repair
without the need' for in vitro culturing of precursor
cells .
It is yet another object of the present invention
to provide a method to enhance the implantability of
bone prosthetic devices.
It is still another object of the present
invention to provide an improved bone implantation
prosthetic device in which the device is seeded with
precursor cells having osteogenic potential isolated
from a patient's peripheral blood, bone marrow, or
adipose tissue.
These and other objects are provided by the
present invention.
The ability to isolate autologous precursor cells
having osteogenic and chondrogenic potential has far
reaching clinical implications for bone and cartilage
repair therapies, either alone or in conjunction with
prosthetic devices. The present invention provides a
simple method for isolating precursor cells having the
potential to generate bone or cartilage from a variety
of tissue types including peripheral blood, marrow, and
adipose tissue. The precursor cells are isolated using
reagents that recognize CD34 or other markers on the
surface of CD34+ precursor cells, for example CD33,
CD38, CD74, and THYl. Significantly, the present
invention does not require in vitro culturing of
isolated precursor cells before the cells can be used
in further in vivo procedures. Indeed, precursor cells
isolated by the present mvention may be transplanted
in vivo immediately for bone or cartilage regeneration.
Thus, the 2 to 3 week time delay required by other
methods for in vitro culturing of progenitor cells is
eliminated making the method economical, practical and
useful for the clinical environment.
Accordingly, the present invention relates to a
method for isolating precursor cells havmg the
potential to generate bone or cartilage directly from
hematopoietic and non-hematopoietic tissues, including
peripheral blood. The method includes steps of
collecting tissue samples, contacting the sample with
an antibody or other reagent that recognizes antigen
CD34 or other antigens on CD34+ precursor cells, and
separating the reagent-precursor cell complex from
unbound material, by for example, affinity
chromatography. Precursor cells isolated by the present
method may be used immediately for bone and cartilage
regeneration in vivo.
In one aspect, the present invention is a method
for isolating precursor cells having the potential to
generate bone or cartilage from peripheral blood,
marrow or adipose tissue.
In another aspect, the present mvention is a
method for isolating precursor cells having the
potential to generate bone or cartilage based on
selecting cells from hematopoietic and non-
hematopoietic tissues that carry cell surface marker
CD34.
In yet another aspect, the present invention is a
method for bone or cartilage regeneration which
utilizes CD34+ precursor cells isolated from peripheral
blood, marrow, or adipose tissue.
DESCRIPTION OF THE PREFERRED EMBODIMENT(S)
Terms used throughout this disclosure are defined
as follows:
Adipose Tissue
A complex tissue containing multiple cell types
including adipocytes and microvascular cells. Adipose
tissue is one of 'the most convenient sources of
precursor cells n the body. As used herein the term
"adipose tissue" is intended to mean fat and other
sources of microvascular tissue in the body such as
placenta or muscle. The term specifically excludes
connective tissues, hematologic tissues, periosteum,
and perichondrium.
Chondrogenic
The capacity to promote cartilage growth. Th s
term is applied to cells which stimulate cartilage
growth, such as chondrocytes, and to cells which
themselves differentiate into chondrocytes. The term
also applies to certain growth factors, such as TGF-β,
which promote cartilage growth.
Connective Tissue
Any of a number of structural tissues in the body
including bone, cartilage, ligament, tendon, meniscus,
and joint capsule.
Differentiat on
A biological process in which primitive,
unspecialized, cells undergo a series of cellular
divisions, giving rise to progeny havmg more
specialized functions. The pathway to terminal
differentiation ends with a highly specialized cell
having unique genetic and phenotypic characteristics.
The conventional wisdom of the past taught that
differentiation proceeded in one direction only - from
less specialized to more specialized. This dogma is now
being challenged by new results which suggest that in
fact the pathway may be bi-directional. Under certain
conditions more specialized cells may in fact produce
progeny which effectively reverse the flow toward
greater specialization.
Hematopoietic Stem Cell
Primitive cell having the capacity to self-renew
and to differentiate into all blood cell types.
Mesenchymal Stem Cell
Primitive cell type having the capacity for self-
regeneration and for differentiating through a series
of separate lineages to produce progeny cells having a
wide variety of different phenotypes, including bone,
cartilage, tendon, ligament, marrow stroma, adipocytes,
dermis, muscle, and connective tissue.
Microvascular Cell
Cells comprising the structure of the microvasculature
such as endothelial, smooth muscle, and pericytes.
Osteogenic
The capacity to promote or to generate the
production of bone. The term may be applied to
osteoblasts which have the capacity to promote bone
growth, or to cells which themselves are able to
differentiate into osteoblasts. The term would also
apply to growth factors having the capacity to promote
bone growth.
Precursor Cell
A cell with the potential to differentiate to
perform a specific function.
Stem Cell
Pluripotent precursor cell having the ability to
self-renew and to generate a variety of differentiated
cell types.
The present invention is premised upon two
surprising discoveries. First, that precursor cells
having the potential to form connective tissue in vivo
can be isolated from a variety of hematopoietic and
non-hematopoietic tissue sources, including peripheral
blood, and adipose tissue. And second, that cell
surface marker CD34, a heretofore unrecognized
identifier for connective tissue precursor cells, may
be used as a marker for precursor cells having the
potential to form bone and cartilage in vivo.
The inventors have discovered two convenient, new
sources for precursor cells (viz. peripheral blood and
adipose tissue) , and a source from marrow which does
not require in vitro culture. Unlike prior methods,
which have used bone marrow or periosteum as the source
for osteogenic and chondrogenic precursor cells, the
present invention enables isolation of these cells from
more conveniently harvested tissues, such as peripheral
blood and adipose tissue. The ability to isolate
osteogenic and chondrogenic precursor cells from
tissues other than marrow and periosteum lends
considerable convenience and simplicity to an otherwise
complicated method.
In one embodiment, the present invention is an
affinity method enabling the isolation of precursor
cells m humans having the potential to generate
connective tissue based on expression of antigen CD34
and other cell surface markers on CD34+ cells. Some
examples of other markers on CD34+ cells would include
CD33, CD38, CD74, and THYl, which list is not intended
to be exclusive. In another embodiment, precursor cells
are isolated from adipose tissue based on differential
sedimentation properties. Significantly, unlike
previous methods, the present invention enables the
immediate use of isolated precursor cells for bone and
cartilage regeneration procedures without the need for
in vitro culturing. As a consequence, the present
method is quicker and easier to implement than
previously described procedures.
I . Isolating Precursor Cells
In one embodiment, the present method for
isolating precursor cells involves collecting a body
tissue sample, contacting the sample with an antibody
or other reagent that recognizes and binds to an
antigen on the surface of the precursor cells, and then
separating the precursor cell-reagent complex from
unbound material by, for example, affinity
chromatography. The method can be applied to peripheral
blood, marrow, or other tissues, including adipose
tissue. For ease and simplicity of isolation, however,
blood is the preferred source material since surgical
procedures are not required,
(a) Peripheral blood as the source of precursor cells
By way of example, about 1 unit of blood is taken
by any suitable means, for example by syringe
withdrawal from the patient's arm. A particularly
attractive method in the clinical environment is
apheresis, which has the added advantage of removing
red cells. Removal of red cells is not essential,
although it does enhance the performance of the method
and is preferred. Red cells may be removed from the
sample by any suitable means, for example, lysis,
centrifugation, or density gradient separation. It is
preferred that the sample also be anticoagulated by,
for example, treatment with citrate, heparin, or EDTA.
The yield of precursor cells is expected to be
about 0.1% to 0.5% of the population of nucleated blood
cells. Yields may vary, depending upon the health and
age of the donor, and on the freshness of the sample.
The yield may be dramatically increased by
administering drugs or growth factors to the patient
before blood collection. Although the method will work
on samples which have been stored under refrigeration,
fresh samples are preferred.
A critical step in the procedure of isolating
precursor cells from peripheral blood involves
contacting the blood sample with a reagent that
recognizes and binds to a cell surface marker on CD34+
cells. Any reagent which recognizes and binds to CD34+
cells is within the scope of the invention. Suitable
reagents include lectins, for example soy bean
agglutinin (SBA) , antibodies and attachment molecules
such as L-selectm.
In the preferred embodiment the sample is
contacted with an antibody against CD34. Either
monoclonal (mAb) or polyclonal antibodies may be used.
Methods for preparing antibodies directed against CD34
and other cell surface antigens on CD34+ cells are well
known to those skilled m the art. Suitable human
antibody preparations directed against CD34 and other
cell surface markers on CD34+ cells may be obtained
commercially from Cell Pro, Inc., Bothell, WA, or
Becton-Dick son, Mountain View, CA.
Suitable cell surface antigens on precursor cells
include CD34 and other antigens on CD34+ cells, for
example THYl, CD33, CD38, and CD74. The preferred cell
surface marker is CD34. It is expected that the
procedure will be successful using other cell surface
antigens on CD34+ cells as markers for precursor cells.
Following a' brief incubation of the sample with
the antibody to enable binding, the precursor cell-
antibody complex is recovered by any suitable method
such as, for example, affinity chromatography, magnetic
beads, and panning. In the preferred embodiment,
recovery is by affinity chromatography. (see, e.g. RJ
Berenson etal . "Positive selection of viable cell
populations using avidin-biotin immunoadsorption" J.
Immunolog. Meth. 91, 11-19, 1986)
Briefly, the affinity recovery method utilizes a
biotin-avidin coupling reaction in which the antibody
is coupled to biotin by any suitable method. The
antibody-biotin labeled precursor cell complex is
separated from unbound materials by passing the
reaction mixture through a column packed with an avidin
labeled matrix. Unbound materials are removed from the
column by washing. A useful commercially available cell
separation kit includes biotin-labeled human anti-CD34
and a column packed with an avidin labeled matrix
("CEPRATE7LC" available from CellPro, Inc. Bothell,
WA) .
Indirect labelling methods are also within the
scope of the invention. For example, the primary
antibody could be directed against a precursor cell
surface marker and a secondary antibody, labelled with
biotin, directed against the primary antibody.
Alternatively, the secondary antibody may be coupled to
a suitable solid support material.
Negative selection schemes are also intended to be
withm the scope of the invention. Using a negative
selection, the antibody, or other reagent, would be
directed against a cell surface marker which is absent
on CD34+ cells.
(b) Bone marrow as the source of precursor cells
The method disclosed above for isolating precursor
cells from blood may be applied m essentially the same
fashion to bone marrow. Bone marrow is collected by any
suitable fashion, for example illiac crest aspiration.
In the preferred embodiment the marrow is treated with
an anticoagulant such as EDTA, heparin, or citrate and
nucleated cells are separated from non-nucleated cells
by any suitable means, for example by hemolysis or by
density gradient centrifugation.
Precursor cells that express the CD34 cell surface
antigen are isolated from marrow using a reagent that
recognizes and binds to CD34 or to some other antigen
on the surface of CD34+ cells. Suitable reagents
include antibodies, lectins, and attachment molecules.
Bound cells are separated from unbound cells by
affinity chromatography, magnetic beads, or by panning.
In the preferred embodiment, an antibody directed
against CD34 is used in the binding reaction and bound
cells are separated from unbound cells by affinity
chromatography, as disclosed more fully in the examples
which follow.
(c) Adipose tissue as the source of precursor cells
As defmed at the beginning of this section,
"adipose tissue" is used throughout this disclosure
a generic sense to mean fat and other tissue types
(excluding connective tissues, hematologic tissues,
periosteum, and perichondrium) which contam
microvascular cells. Microvascular tissue, from which
capillaries are made, is an integral part of the blood
transport system and, as such, is ubiquitous throughout
the body. Microvascular tissue is composed of at least
three cell types - endothelial, pericytes, and smooth
muscle. Early investigations suggested that
microvascular tissue might play an important role in
bone metabolism. "A key observation was that
microvascular cells and tissue arose de novo and
proliferated at sites of bone repair and new bone
growth. Such observations led to speculation that
endothelial cells, pericytes, or both may be
osteoprecursor cells, or alternatively, that
microvascular cells exert a mitogenic effect on bone
precursor cells. (See e.g. C Brighton et.al. "The
pericyte as a possible osteoblast progenitor cell"
Clin. Orthop. 275, 287-299, 1992) A more recent study
using in vitro cultured cells suggests both progenitor¬
like cell proliferation and mitogenic effects by
microvascular cells. (AR Jones et.al. "Microvessel
endothelial cells and pericytes increase proliferation
and repress osteoblast phenotype markers in rat
calvarial bone cell cultures" J. Ortho. Res. 13, 553-
561, 1995) . Thus, within the microvascular cell
population are precursor cells having osteogenic and
chondrogenic potential.
The method of the present invention, as applied to
adipose tissue, has two embodiments. In the first
embodiment, the tissue is contacted with a reagent that
recognizes CD34 or other surface antigen on CD34+
cells. As with peripheral blood and marrow, suitable
binding reagents for use with adipose tissue include
lectins, antibodies, and attachment molecules. The
affinity binding method, as applied to adipose tissue,
differs from the method as applied to blood and marrow
by requiring a step to produce a single-cell suspension
before incubation with the antigen binding reagent. Any
suitable dissociation enzyme such as, for example,
collagenase may be used. Cells that bind the reagent
can be removed from unbound cells by any suitable
means, for example affinity chromatography, magnetic
beads, or panning.
In the preferred embodiment of the invention as
applied to adipose tissue, a sedimentation method is
utilized to obtain a fraction of cells that is enriched
for precursor cells having osteogenic and chrondrogenic
potential. Following harvest of the tissue and
digestion with an enzyme to form a single-cell
suspension, the cells are separated by gravity
sedimentation on the bench top, or by centrifugation.
By way of example, fat could be secured by
liposuction or any other suitable method. About 10 cc
to 30 cc of fat tissue is digested with enough
dissociation enzyme (e.g. collagenase) to produce a
single-cell suspension. Suitable reaction conditions
for enzyme digestion will vary depending on the enzyme
used, as known to those skilled in the art. Following
enzyme digestion, the adipocytes are separated from
other cell types by centrifugation. Adipocytes float to
the surface while denser cells, which include precursor
cells, collect on the bottom and are separable
thereafter by any suitable means. After washing the
harvested precursor cells they can be mixed with a
suitable carrier and immediately implanted in vivo at a
site needing repair.
II. In Vivo Mesenchymal Tissue Regeneration
The precursor cells recovered by the present
procedure are useful for a variety of clinical
applications. For example, they may be transplanted
without further processing to a connective tissue site
in a patient to promote the repair or regeneration of
damaged bone or cartilage.
Unlike previous methods, the present invention
does not require in vitro culturing in order to obtain
a suitable cell type or an adequate quantity of
precursor cells to be of use for in vivo application.
The present invention takes advantage of the unexpected
finding that osteogenic and chondrogenic precursor
cells may be isolated from a variety of hematopoietic
and non-hematopoietic body tissues such as peripheral
blood and adipose tissue. This finding has created a
heretofore unappreciated reservoir of precursor cells
that can be drawn from conveniently to provide enough
cells for in vivo applications without an additional
time-consuming step of amplifying cell numbers by in
vitro culturing. This aspect of the invention saves
time and money with less risk of complication and pain
for the patient.
By way of example only and in no way as a
limitation on the invention, the precursor cells
isolated by the present method from any suitable tissue
source may be implanted at any connective tissue site
needing bone or cartilage regeneration. Suitable
implanting procedures include surgery or arthroscopic
injection.
While the factors that determine biological
differentiation are not fully understood, it is known
that precursor cells will differentiate into bone or
cartilage if transplanted to a site in the body needing
repair. Precursor cells isolated by the present method
can be implanted alone or premixed with growth factors
such as TGF-β. It is preferred that the cells be mixed
with a suitable carrier material, well known to those
skilled in the art, so as to impede the dislodgement of
implanted cells. A non-exclusive list of suitable
carriers would include, for example, proteins such as
collagen or gelatin; carbohydrates such as starch,
polysaccharides, saccharides, methylcellulose, agar, or
algenate; proteoglycans; synthetic polymers; ceramics;
or calcium phosphate.
The data presented in Table 2 demonstrate the
operability of the invention for in vivo applications.
The rat calvarial model used in these studies
demonstrated that CD34+ cells isolated from marrow
using a monoclonal antibody were as effective at
promoting bone growth in an in vivo environment as were
the positive controls (autologous graft) . The data also
show that the antibody itself can affect the outcome of
the results probably via interaction with the
complement system. For example, cells bound by mAb 5E6
did not stimulate bone growth in the rat calvarial
model. Although both antibodies tested recognize CD34
and are IgM isotypes, 5E6 binds complement effectively
while 2C6 does not.
III . Prosthetic Devices
A variety of clinically useful prosthetic devices
have been developed for use in bone and cartilage
grafting procedures, (see e.g. Bone Grafts and Bone
Substitutions. Ed. M.B.Habal & A.H. Reddi, W.B.
Saunders Co., 1992) For example, effective knee and hip
replacement devices have been and continue to be widely
used m the clinical environment. Many of these devices
are fabricated using a variety of inorganic materials
having low immunogenic activity, which safely function
in the body. Examples of synthetic materials which have
been tried and proven include titanium alloys, calcium
phosphate, ceramic hyroxyapatite, and a variety of
stainless steel and cobalt-chrome alloys. These
materials provide structural support and can form a
scaffolding into which host vascularization and cell
migration can occur.
Although surface-textured prosthetic devices are
effectively anchored into a host as bare inorganic
33 structures, their attachment may be improved by seeding
with osteogenic precursor cells, or growth factors
which attract and activate bone forming cells. Such
"biological-seeding" is thought to enhance the
effectiveness and speed with which attachment occurs by
providing a fertile environment into which host
vascularization and cell migration can occur.
The present invention provides a source of
precursor cells which may be used to "seed" such
prosthetic devices. In the prefered embodiment
precursor cells are first mixed with a carrier material
before application to a device. Suitable carriers well
known to those skilled in the art include, but are not
limited to, gelatin, collagen, starch, polysaccharides,
saccharides, proteoglycans, synthetic polymers, calcium
phosphate, or ceramics. The carrier insures that the
cells are retained on the porous surface of the implant
device for a useful time period.
A more complete understanding of the present
invention can be obtained by referring to the following
illustrative examples of the practice of the invention,
which examples are not intended, however, to be unduly
limitative of the invention.
EXAMPLE 1
Animal model for bone regenera ting capaci ty of
precursor cells
A rat calvarial model was used to test the
operability of the invention for in vivo applications.
The model consisted of monitoring the ability of
various test samples to promote bone growth in
calvarial defects which had been surgically introduced
into the rat skull. Calvarial defects were introduced
into 6 month to 9 month old Fisher rats having
bodyweights in the range of about 300 g to 500 g
according to the following procedure. Animals were
anesthesized by intramuscular injection using a
Ketamine- Rompun (xylazine)- Acepromazine (acepromazine
maleate) cocktail, and surgical incisions made in the
calvarial portion of the skull. After peeling back the
skin flap, a circular portion of the skull measuring 8
mm in diameter was removed using a drill with a
circular trephine and saline irrigation. An 8 mm
diameter disk of "GELFILM" was placed in each defect to
separate the exposed brain from the test material and
to maintain hemostasis. The calvarial defects produced
in this fashion were then packed with a test sample
consisting of an isolated cell population. For some
experiments the test samples were mixed with a carrier
material consisting of rat tail collagen or "AVITENE"
bovine collagen before introduction into the calvarial
defect. The positive control consisted of an autograft
while the negative control consisted of a tricalcium
phosphate (TCP) carrier only implant. After surgical
closure of the wound site, treated animals were
returned to their cages, maintained on a normal food
and water regime, and sacrificed 28 days after surgery.
The effectiveness of a test sample to induce bone
growth in calvarial defects was assessed by estimating
new bone formation at the site of the defect by
measuring the closure in the linear distance between
cut bone edges or noting islands of bone growth in the
central portion of the defect. The scoring criteria are
shown in Table 1. The results are summarized in Table
2.
EXAMPLE 2
Isola tion of an enriched nuclea ted cell popula tion from
ra t bone marrow.
Rat bone marrow was isolated from the
intramedullary cavities of 6 femurs taken from male
Fisher rats between 8 to 10 weeks of age. Prior to
sacrifice the animals had been maintained on a normal
food and water diet. The marrow was extracted from
excised femurs by flushing into a test tube containing
approximately 5 ml of ACD buffer. Buffer ACD in the
neat state consists of 2.2g Na3Citrate.2H20, 0.8g
citric acid, and 2.4g dextrose dissolved in 100 ml
distilled water. Unless otherwise noted, buffer ACD was
diluted to a concentration of 15% in PBS. The extracted
marrow cells were gently suspended into the buffer
solution by pipetting. In order to separate red cells
from white cells, the marrow cell suspension was
underlaid with approximately 4 ml of Ficoll-Hypaque
with a specific gravity of 1.09 (Sigma Chemical Co.,
St.Louis, MO) and centrifuged at 1200 x g for 20
minutes. After centrifugation the interface layer
containing the nucleated cells was removed by
pipetting. The cells were washed in 5 ml of ACD and
centrifuged at 250 x g for 6 to 7 minutes. The pellet
was washed twice more in 1% BSA/PBS (bovine serum
albumin, phosphate buffered saline; supplied with
CEPRATE LC kit) /All PBS was Ca+2 and Mg+2 free to
prevent clotting.
EXAMPLE 3
Isola tion of CD34+ cel ls from ra t bone marrow using a
monocl onal an tibody and affini ty chroma tography and
their use for in vivo bone regenera tion in ra t
ca lvarial model .
Ma terials and Methods .
Mouse IgM monoclonal antibodies 2C6 and 5E6 were
raised against rat CD34 present on the surface of a
subpopulation of rat hematopoietic cells. The CD34
mAb' s used in these experiments were the gift of Dr.
Othmar Forster and were prepared in a manner well-known
to those skilled in the art. Anti-mouse IgM:FITC, used
for fluorescence sorting of cells bound with mAb's 2C6
and 5E6, was obtained from Boehringer Mannheim, Cat. #
100807. Avidin: FITC also used in fluorescence sorting
was obtained from Boehringer Mannheim, Cat. # 100205.
CD34+ cells labeled with mAb 2C6 or 5E6 were separated
from unbound cells using an affinity column method. A
useful, commercially available affinity cell separation
kit, "CEPRATE LC, " may be obtained from CellPro
(CellPro, Inc. Bothell, WA 98021) . Anti-mouse
IgM:biotin was purchased from Southern Biotech,
Birmingham, AL, Cat. # 1022-08.
Cells carrying the CD34 surface antigen were
isolated from rat marrow as follows. The rinsed
nucleated cells, isolated in the manner described in
Example 2, were resuspended in about 0.5 ml of
1%BSA/PBS (from CellPro kit) . Then, a volume of mAb
ranging in concentration from about 1 μg/ml to 40 μg/ml
was added and the mixture incubated for about 1 hour at
room temperature with occasional, gentle agitation.
Following incubation the mixture was brought to 10 ml
with 1%BSA/PBS and the mixture centrifuged at 250 x g
for 6 minutes. The pellet was gently resuspended and
rinsed two additional times in 10 ml 1%BSA/PBS and spun
as before. After another resuspension and
centrifugation, the final cell pellet was resuspended
in 2 ml 1%BSA/PBS for incubation with a biotinylated
anti-mouse IgM.
About 10 μl of Goat anti-mouse IgM:biotin (0.5
mg/ml before dilution) was added to the resupended mAb-
cell pellet obtained at the previous step. The mixture
was incubated at room temperature for about 30 minutes
with gentle agitation, after which the cells were
rinsed twice by centrifugation and resuspension in
BSA/PBS, as previously described. The final cell pellet
was resuspended to about 100 x 106 cells/ml in 5% BSA
in a volume of 1 ml to 4 ml for loading onto an avidin
column.
Antibody-labeled and unlabeled cells were
separated on the "CEPRATE LC" avidin column using the
conditions recommended by the manufacturer (Cell Pro,
Inc., Bothell, WA) . Briefly, the column contained a bed
of PBS- equilibrated avidin matrix. Prior to loading
the sample, about 5 ml of 5% BSA was run through the
column. The pre-diluted cell sample was then layered
onto the top of the gel matrix and the sample
thereafter allowed to run into the matrix gel.
Unlabeled cells were washed from the column with about
3 ml to 5 ml of PBS. The mAb-labeled cells were then
released from the matrix and collected into a small
volume of 5% BSA by gently squeezing the column so as
to agitate the matrix while washing the column with
PBS. Small aliquots were saved from the bound and
unbound fractions for cell counting and flow cytometry.
For implantation experiments the cells were washed 2
times in PBS/BSA and once in PBS only.
.Resul ts .
Each experiment generated about 10 to 20 x 106
adherent cells of which about half this number were
implanted into a calvarial defect. Cell fractions taken
from the column were tested for viability by trypan
blue cell counts using a hemacytometer and found to be
in the range of about 85% to 97% viable. The adherent
cell population appeared to be a group of small blast
cells. FACS was used to determine the purity of CD34+
cells isolated on the column. The adherent cell
population contained about 50% of the original number
of CD34+ cells at a purity of about 50%.
CD34+ cells were implanted into rat calvarial
defects with or without a suitable carrier material.
Two carriers were tried in these experiments, "AVITENE"
and rat tail collagen, both of which were found to be
useful. Rat tail collagen is preferred, however, since
it showed the least inflammatory response. About 50 mg
of collagen was dissolved in 1 ml of PBS at 60°C and
equilibrated to 37°C prior to mixing with cells. In
some experiments pellets containing collagen and cells
were formed by mixing 100 μl of collagen solution with
a cell pellet and cooling the mixture to 4°C prior to
implantation mto a calvarial defect. Surgical
implantations were performed as described in Example 1
with sacrifice of recipient animals at 28 days post-
surgery.
Histology scoring for bone formation was assessed
according to the scheme shown in Table 1.
Discussion .
The finding that CD34+ cells isolated by mAb 5E6
failed to stimulate bone regeneration vivo may be
explained by the ancillary observation that this
antibody is a more effective activator of the
complement system than mAb 2C6 (data not shown) .
EXAMPLE 4
(a) Bone regenera tion m ra t calvaria l model using
Fi coll -separa ted whole blood.
The rat calvarial model described in Example 1 was
used to determine the bone regenerating capacity of
Ficoll-separated whole blood. Approximately 2.5 ml of
donor blood was used for each recipient calvarial
defect. Donor animals were 8 to 10 week old male F344
strain rats. Recipients were 6 to 8 months old. Donors
were bled into 3 cc syringes, which contained about 0.5
cc of ACD solution to inhibit coagulation.
ACD Stock Solution ACD Working Solution
2.2 g Na3Citrate.2H2015 ml ACD Stock Solution
0.8 g citric acid.lH2θ 100 ml PBS (Ca++/Mg++ free)
2.4 g dextrose
100 ml distilled water
Blood was placed into 15 ml conical tubes and
brought up to 5 ml with ACD working solution. The
samples were underlaid with 4 ml of Ficoll-Hypaque and
centrifuged at 1200 xg at room temperature for 20
minutes. After centrifugation, the white cell layer was
removed from each tube by pipet.
Ficoll-separated blood cells were used for
implantation experiments, either directly or after
mixing with a carrier material. For direct
implantation, the cell pellet was washed twice in 10 ml
of PBS and the final pellet, containing roughly 5 to 10
x 106 cells, delivered neat into a calvarial defect.
Cell samples pre-mixed with a carrier material were
combined with rat tail collagen prior to implantation.
About 50 mg of rat tail collagen (obtained from Sigma,
St. Louis, MO; Cat.# C-8897) was heated to 60°C in 500
μl PBS to dissolve the collagen protein. The collagen
solution was equilibrated to 37°C prior to mixing with
the cell pellet. About 60μl of collagen solution was
mixed with the cell pellet and the entire cell-collagen
mixture implanted into a calvarial defect.
EXAMPLE 5
Isola tion of CD34+ cells from ra t blood using a
monoclonal an tibody and affini ty chroma tography.
(1) Hemolysis Buffer - IPX Stock Solution
Dissolve the following in 1 L distilled water, adjust
pH to 7.3, filter sterilize and store at 2 - 8°C.
83 g NH4C1
10 g NaHC03
4 g Na2EDTA
(2) Phosphate Buffered Saline (PBS) Ca2+ and Mq2+
Free
Dissolve in 1 L distilled water, adjust pH to 7.2,
filter sterilize, and store at 2 - 8°C.
8 g NaCl
1.15 g Na2HP04
0.2 g KH3P04
0 . 2 g KCl
(3) PBS + Bovine Serum Albumin
Dissolve lg BSA in 100 ml PBS.
(a) Approximately 100 ml of whole blood was
collected by cardiac puncture from 17 male F344 rats 8
to 10 weeks old and heparinized by standard procedures.
Red cells were lysed by mixing the whole blood with 300
ml of IX hemolysis buffer at 37°C and allowing the
mixture to sit for about 3 minutes. Then 100 ml of
PBS/BSA washing solution was added and the mixture
centrifuged at 170 xg for 10 minutes. The resulting
supernatant was aspirated without disturbing the cell
pellet. The pellet was washed two more times by gently
resuspending in PBS/BSA followed by centrifugation. The
final pellet was brought up to 2 ml in PBS/BSA in
preparation for incubation with the mAb, and a small
aliquot removed for cell counting and FACS analysis,
(b) The cell pellet, resuspended in 2 ml PBS/BSA
as in step (a) , was incubated with 3 ml of neat mAb 2C6
in order to bind CD34+ cells. The mAb-cell mixture was
incubated at 4°C for 45 minutes and the cells gently
agitated once to resuspend during incubation. Following
the incubation period the volume was brought up to 10
ml with PBS/BSA and the sample washed twice as in step
(a) . The washed pellet was resuspended in 2 ml PBS/BSA
and 15 ml of goat anti-mouse IgMrbiotin was added for a
30 minute incubation at 4°C with one gentle agitation
during incubation to resuspend cells. The cells were
rinsed twice in PBS/BSA, as described in step (a) , and
the final pellet resuspended in 10 ml of 5% BSA. 5 ml
of the resuspended pellet were used for each of two
"CEPRATE LC" column sorts, as described in Example 3.
Antibody-bound cells were released from the column as
described in Example 3 and the released cells washed
twice in PBS/BSA, and once in PBS. The final cell
pellet was mixed on a glass slide with 60ml of rat tail
collagen (100 mg/ml) at 37°C, and the mixture of
collagen and cells placed briefly on ice to form a
solid pellet. The cell-containing pellet was then
transplanted immediately into a rat calvarial defect,
as described in Example 1.
EXAMPLE 6
Isola tion of microvascular cells from ra t epididymal
fa t pads .
Two epididymal fat pads were removed by dissection
from a male Fisher F344 rat, minced with scissors under
sterile conditions, and incubated in 10 ml PBS/1%BSA
the presence of 8 mg/ml collagenase (Type II Crude,
273U/mg; Worthington Laboratories) for 45 minutes at
37°C with gentle shaking. After digestion the sample
was centrifuged at 250 xg for 4 minutes and the low
density fat at the top of the tube removed by
aspiration. The pellet, which contained the precursor
cells, was washed twice in PBS/1%BSA and once in PBS.
The washed pellet was mixed with 50 ml rat tail
collagen at 37°C, placed briefly on ice to gel, and
implanted into a rat calvarial defect.
It is thought that the method for isolating and
using bone and cartilage precursor cells by the present
invention and many of its attendant advantages will be
understood from the foregoing description and it will
be apparent that various changes may be made in the
form, construction, and arrangement of the elements
thereof without departing from the spirit and scope of
the invention or sacrificing all of ts material
advantages, the form hereinbefore described being
merely a preferred or exemplary embodiment thereof.
Table 1
Bone Formation Scoring
Site Score Description
Defect 0 No net gain in bone; either less
formation than resorption or no
formation at all.
1 Less that 5% of linear distance
between cut bone edges is bridged by
new bone.
2 About 5% to 33% of the defect is
bridged by new bone, or there is an
island of bone in the central portion
of the defect.
3 About 33% to 66% of the defect is
bridged by new bone.
4 Greater than 66% of the defect is
bridged by new bone.
5 Complete bridging of the defect by new
bone.
Table 2.
RBRA Tissue/Cell Type N (Mean ± S.D.)
Autologous Graft (positive control) 1 14422 2.4± 0.7
TCP (negative control) 105 l.O± 0.9
Marrow 30 2.5± 1.1
Marrow Ficoll 18 2.3± 0.8 Marrow/Avitene 9 1.8± 0.4
Blood Ficoll 11 1.3± 0.5
Blood/RTC Ficoll 16 1.4+ 0.5
2C6+ cells 12 1.8± 0.4
2C6- cells 12 0.7± 0.5 5E6+ cells 12 1.3+ 0.6
5E6- cells 12 1.5+ 0.5
SBA+ cells 12 l.β±l.l
SBA- cells 18 1.4+0.7
RBRA: Relative bone regeneration activity
N: Number of experiments
S.D.: Standard deviation
2C6 and 5E6 cells were isolated from marrow
SBA: Soy Bean Agglutinin