WO2000022097A2

WO2000022097A2 - Periferal blood mensenchymal precursor cells

Info

Publication number: WO2000022097A2
Application number: PCT/GB1999/003402
Authority: WO
Inventors: Nathan Joseph Zvaifler; Lilia Marinova-Mutafchieva; Ravinder Nath Maini
Original assignee: The Mathilda And Terence Kennedy Institute Of Rheumatology
Priority date: 1998-10-14
Filing date: 1999-10-14
Publication date: 2000-04-20
Also published as: WO2000022097A3; EP1135464A2; GB9822434D0

Abstract

Isolated PBMPC's are described. Such PBMPC's behave like mesenchymal stem cells in that they lack differentiation markers and may be stimulated to differentiate into different types of cells. PBMPC's are obtainable by elutriating human blood. Such PBMPC's may be used for gene therapy or tissue engineering.

Description

PERIFERAL BLOOD MESENCHYMAL PRECURSOR CELLS

The invention relates to cells isolated from blood, hereinafter referred to as (PBMPC)

periferal blood-derived mesenchymal precursor cells (PBMPC), their charaacteristics,

methods for their isolation, and to their use.

Bone marrow is a complex tissue containing progenitors of haematopoeitic cells, their

progeny, and a connective tissue network of mesenchymally derived cells, known as

stroma. The stroma contains cells that can differentiate into bone, cartilage, fat, muscle,

and a reticulum which supports haematopoeitic stem cell differentiation (1,2).

Extensive experimentation has defined in vitro and in vivo conditions for the isolation,

propagation and differentiation of these cells, which are referred to as marrow

mesenchymal stem cells (MMSC). These are a population of cells that firmly adhere to

tissue culture vessels, lack differentiation markers, have a high proliferative capacity

and have the potential for self renewal and multipotentialiry when appropriately

stimulated. Similar cells have not previously been demonstrated in the circulation of

normal individuals.

Stromal stem cells arise from embryonic mesenchyme, which in turn is derived from

the neural crest and mesoderm of the developing embryo. Appendages known as limb

buds, are essentially composed of mesoderm covered by ectoderm. The mesenchyme

condenses and gives rise to bone and cartilage. Cavitation within the condensed

mesenchyme is the site of the future joint. This process can be followed using

antibodies to bone morphogenetic protein (BMP) receptors (Ishidou, Y.J. Bone Mineral Res 10:1651, 1995). BMPs are a group of multifunctional proteins, related to the TGF

beta family, that induce proteoglycan synthesis in chondroblasts, alkaline phosphatase

activity, the cAMP response to parathyroid hormone, type 1 collagen synthesis in

osteoblasts, and differentiation of neural cells. BMPSs can cause ectopic bone

formation in vivo and also play a critical role in morphogenesis.

Cells that assume features of fibroblasts, from differentiating mesenchymal cells, have

been derived from blood previously. Fernandez (7) described low density mononuclear

cells in the peripheral blood of patients with breat cancer mobilised by a growth factor,

i.e. granulocyte-colony stimulating factor (G-CSF), which, following one week of tissue

culture grew into adherent fibroblast-like cells, whilst a few cells appeared large, flat

and round. Immunohistology and flow cytometric analysis (FACS) revealed fibronectin

and several types of collagen (I, III, VI) in the cytoplasm of cultured cells. The cells

expressed adhesion ligands and antigens recognised by a pair of monoclonal antibodies

(SH2 and SH3) purported to be MMSC markers (1). Such cells were not demonstrated

in the peripheral blood of normal individuals who had not been treated with G-CSF (7).

Moreover the capacity of pluripotential differentiation was not demonstrated. Bacala

separated human blood cells by density centrifugation, cultured them on a fibronectin

matrix, and identified a population of circulating cells with fibroblast properties and a

distinctive phenotype (collagen+/CD34+). This novel cell termed a fibrocyte, has both

mesenchymal and hematopoietic features (8). Since specific phenotypic makers of

bone marrow MMSC are not well characterised, the relationship of the cells hitherto

isolated from blood kto bone marrow MMSC is unknown. The inventors have demonstrated for the first time that cells with the morphology and

phenotype of mesenchymal progenitor (stem) cells described in bone marrow are

normally present in the circulation. The observations that support these conclusions

and the significance of the findings are discussed below. The inventors have

unexpectedly identified cells from human blood which have the physiological

characteristics and differentiation potential tuypical of bone marrow mesenchymal cells.

The lack of definitive and specific phenotypic markers at present does not permit the

conclusion that the cells derived from blood are the one and the same as those present

in the marrow. For this reason we have used the term peripheral blood derived

mesenchymal precursor cells (PBMPC) to describe the cells present in the blood.

The identification that such cells are obtainable from blood means that isolation of

PBMPC is substantially easier than obtaining them from bone marrow.

Such PBMPC cells may be used for experimentation to follow their differentiation into

other forms of cells. They may also be used in the regeneration of bone, cartilage,

fibrous tissues such as ligaments and tendons, muscle or other connective tissues

damaged by acute injury, genetic or acquired diseases. Furthermore, such precursor

cells are ideal vehicles for gene therapy.

A first aspect of the invention provides an isolated periferal blood-derived

mesenchymal precursor cell (PBMPC). PBMPC's are small, round cells which are mononuclear cell-like in their appearance.

They are not depleted with antibodies against the CD34 marker. They have the

potential for self-renewal and multipotentiality when appropriately stimulated. They

lack haematopoietic markers and lymphocytic markers such as CD 14, HLA Class II,

CD45, CD20, CD34 and CD3.

PBMPC's are isolated from mammalian blood, especially human blood.

Preferably, the PBMPC's are obtainable by elutriating human blood.

The invention also provides a method of isolating an PBMPC comprising elutriating a

sample of mammalian blood.

Preferably the sample of blood is a sample of anti-coagulated platelet-depleted

leucocytes, known as "buffy coats".

The sample of blood is preferably depleted of red blood cells. This may be carried out

using techniques known in the art, such as density gradient cell separation.

Preferably centrifugal elutriation is used to isolate the PBMPC. Typically, a suspension

of cells is pumped into a funnel shaped chamber at preset flow rate. As fluid travels

through the chamber its velocity decreases as the chamber gets wider, thus creating a

velocity gradient from the narrow end of the chamber to its widest part. Cells migrate

to positions in the velocity gradient where the effects of both the centrifugal force field and the fluid velocity are balanced. Smaller cells are at equilibrium at the elutriation boundary where the centrifugal force field and the velocity are low.

The cells obtained from the separation step, such as the elutriation step, may be washed with a suitable buffer such as FCS-DMEM. The cells may then be cultivated by cell

culture.

Preferably the elutriator is spun at between 10,000 RPM and 30,000 RPM, preferably

25,000 RPM. The elutriator may be chilled to, for example, 10°C.

The flow rate through the elutriator is preferably 10 to 16 ml./min., especially 12

ml./min.

Flow rates and spin rates may vary depending on the make of the elutriator used. However, optimisation of the flow rates and spin rates may be carried out by those

skilled in the art without undue experimentation.

Aliquots from the elutriation step may be selected from the fractions with the majority

of PBMPC. The PBMPC typically come out of the elutriator in the same aliquots as those containing between 50% and 75% monocytes of the total of cells within the aliquot. Typically, PBMPC are concentrated after between 1200 and 1500 ml. of elutriation buffer has been collected from the elutriator. The isolated PBMPC may be centrifuged. washed, and plated into suitable growth

medium. This allows the PBMPC to grow and to begin to differentiate.

Preferably the growth medium used is complete growth medium (DMEM plus 20%

sterile heat inactivated FCS). This favours fibroblast growth. Alternatively, the

PBMPC are cultured in complete growth medium supplemented with dexamethasone,

ascorbic acid and β-glycerophosphate, this supports the growth of osteoblast-like cells

and adipocyte-like cells.

Accordingly, the invention also includes within its scope a method of producing

differentiated cells comprising the steps of: providing periferal blood mesenchymal

precursor cells, which may have been obtained by a process according to the first aspect

of the invention, and culturing the cells under predefined cell tissue culture conditions

to produce differentiated cells for tissue engineering.

Periferal blood mesenchymal precursor cells according to the invention may also be

used as vehicles for gene therapy. They are simple to obtain, and can be expanded in

vitro, prior to transfection with a gene of interest. The periferal blood mesenchymal

precursor cells can then be infused systemically to return them to the bone marrow or

provide progeny for the repopulation of other tissues (Prockop, D., Science, Vol. 276,

page 71, 1997).

A principal advantage of the periferal blood mesenchymal precursor cells over, for

example, bone marrow-derived cells is that they can be readily obtained from blood. Furthermore, the use of elutriation allows sufficient yields of the cells to be isolated and

propagated. This is especially useful for gene therapy, where large concentrations of

cells may be required. The periferal blood mesenchymal precursor cells obtained also

have the advantage that they replicate quickly. Typical doubling times of 2.5 - 3 days

have been observed by the inventors.

The invention will now be described by way of example only, with reference to the

following figures:

Figure 1

Appearance of cells isolated from an PBMPC-rich elutriation fraction of healthy human

blood and cultured in DMEM-20% FCS for 8 days. The predominant cells are both

fibroblast-like with a central nucleus, filmy cytoplasm and adherent pseudopods and

large round cells with a thin adherent cytoplasm and a round central nucleus.

Hematoxylin (A) 200X-left; and (B) 400X-centre. On the right (C) are the same cells

isolated from an PBMPC-rich elutriation fraction of healthy human blood but cultured

in DMEM-20%) FCS supplemented at the initiation of culture with dexamethasone

(10-⁷M), ascorbic acid 2-phosρhate (0.05 mM) and β-glycerophosphate (lOmM) for 8

days. At this time all the cells have a round or cuboidal morphology with a centrally

placed nucleus (200X). Compare with non supplemented cultures in A.

Figure 2

Time lapse video cinematography of an PBMPC enriched elutriation fraction cultured

in DMEM-20% FCS observed by phase contrast microscopy. At Day 2 (left) there are cells in clusters and occasional cells with pseudopods. By Day 6 there are many large

fibroblast-like cells and large round stromal cells arising from a cluster of small round

cells.

Figure 3

An PBMPC-rich elutriation fraction from healthy human blood cultured in

DMEM-20% FCS. Cells were fixed, stained with anti-BMPR 1A antibody, and

analysed on days 3, 5, 8 and 11. PBMPC were determined by morphology

(fibroblast-like - hatched bars - or big cells - filled bars) and immunoperoxidase

staining. Results are presented as the mean of total number of cells in six individual

images (400X-computer image analysis - AnalySIS).

Figure 4

PBMPC cultured in DMEM-20% FCS supplemented with dexamethasone, ascorbic

acid and β glycerophosphate (described in Figure 1) (A). Day 10 there are many large

multinucleated cells (phase contrast microscopy) that stain with antibody to the

vitronectin receptor (αvβ3) (B). Day 20 cultures have many large cells (3 to 6 times

bigger than PBMPC). Most large cells stain with an antibody to osteocalcin (C). Some

large cells in the supplemented cultures contain fat (adipocytes) and stain with Sudan

IV dye. Figure 5

PBMPC-rich elutriation fractions (n=4) cultured in DMEM-20% FCS with varying

concentrations of BMP-2 for 5 days. Supematants were collected and analysed for

alkaline phosphatase activity (AP). The lowest concentration of BMP-2 (1 ng/ml.)

caused a significant increase in AP activity. **=p0.004.

Figure 6

Gene expression for (A) the chemokine SDF-1 or (B) the housekeeping gene

glyceraldehyde-3 -phosphate dehydrogenase (GA3PD) in cultured circulating PBMPC

(GA42, GA43), a rheumatoid arthritis synovial fibroblast line (RA-505 passage 4), and

an SDF-1 plasmid. RT-PCR and the primers used are described in Methods.

MATERIALS AND METHODS

Reagents

The following reagents were purchased from Sigma (U.K.): dexamethasone, ascorbic

acid-2 phosphate, β-glycerophosphate, bovine serum albumin (BSA), and fetal calf

serum (FCS); penicillin, streptomycin, DMEM and RPMI-16% from Biowhittaker

(Watersville, MD.); Lymphoprep (Nycomed Oslo, Norway).

Monoclonal antibodies were purchased from commercial vendors unless otherwise

stated: CD3, IgGl; CD68, IgGl; CD34, IgGl ; CD45, IgGl and IgG2a controls (Dako

Corp., Carpinteria, CA.); anti-HLA-DR, IgG2a; CD14, IgG2b; CD34, IgGl (Becton

Dickinson, San Jose, CA); anti-vimentin, IgGl ; IgG2b control (Serotec, Oxfordshire,

U.K.); anti-NCAM-1, IgGl (Pharmingen, San Diego, CA.), anti-collagen type 1, IgGl

(Sigma, St. Louis, MO), anti-osteocalcin, IgGl (HaemTech, Essex, Jet, NT), and

anti-αvβ3 (vitronectin receptor) IgGl. Stro-1 is a culture supernatant, monoclonal IgM,

from Developmental Studies Hybridoma Bank, University of Iowa (Iowa City, IA).

Biotinylated mouse Ig, streptavidin-HRP conjugate, DAB, and Nectastain ABC (Vector, Burlingame, CA). Tissue Culture Plates and Glassware

"Culture slides" - Tissue culture treated glass slides; Petri dishes; 6 well tissue culture

plates (Falcon, Becton Dickinson Labware, Franklin Lakes, N.J.); 12 well sterile glass slides (I.C.N., Costa Mesa, CA).

Elutriation Procedure for PBMPC

Anti-coagulated, platelet deplete, buffy coats are obtained as sterile packages from the

North London Blood Transfusion Service. The buffy coat, approximately 50 ml., is

diluted one to four in RPMI and 25 ml. is layered over 20 ml. of Lymphoprep™ in a 50

ml. conical centrifuge tube.

The tubes, (approximately 8 in number) are centrifuged at 2000 rpm for 20 minutes.

The supematent is discarded and leukocyte-rich interface cells collected and combined.

These are made up to 20 ml. in RPMI and layered again over Lymphoprep™ and

centrifuged at 2000 rpm. for an additional 20 minutes. The buffy coat, now depleted of

red blood cells, is collected from the interface, resuspended in 50 ml. of sterile RMPI

and 5% heat inactivated FCS and introduced into the sample line of the flow system of

a Beckman JE-50 cell elutriator which has been charged with elutriation buffer. The

chamber is centrifuged at 25,000 rpm. at 10°C and the flow rate adjusted to 12 ml./min.

The eluate fractions are collected in sterile conical 50 ml. tubes. After collecting

approximately 150 ml. the flow rate is increased by 1 ml./min. The fractions #1-6 (flow

rates of 12-16 ml./min.) contain the bulk of the lymphocytes. Monocytes usually make their appearance in fractions 6 or 7 (determined by FACS analysis). In fraction 7 or 8

monocytes constitute up to two-thirds of the cells and PBMPC are concentrated in these

fractions. Elutriation fractions containing more than 50%> and less than 15% monocytes

are centrifuged at 1200 rpm. for 5 min. The cell pellets are combined, reconstituted in

DMEM plus 20% sterile heat inactivated FCS (hereafter referred to as complete

medium, unless otherwise stated), counted in a haemocytometer, washed in medium

and pelleted, resuspended to 5 x lOVml., and dispensed into either tissue culture plastic

or glass chamber slides. More than 100 consecutive buffy coats from normal

individuals were processed by this method and cultured In every case the appropriate

elutriation fractions had cells with the typical PBMPC morphology.

Cell Proliferation Assay

The PBMPC-rich elutriation fractions are plated at 5 x 10⁵ in 500 μl of complete

medium in polystyrene chambers on tissue culture treated glass slides (Falcon). At

various times cultures are rinsed twice with Tyrode's balanced salt solution, fixed with

1%) glutaraldehyde (v/v) in Tyrode's for 15 minutes, rinsed twice with deionized water,

and air-dried. Cultures are then stained with 0.1 % crystal violet (w/v) in deionized

water for 30 minutes, washed 3 times with deionized water, crystal violet dye is

extracted by rocking gently in 1%> Triton XI 00 for 4 hours at room temperature and

read at 595 nm on a microplate reader (BioRad). Absorbance values (OD) are

converted into absolute cell numbers based on established standard curves (9). Immunohistochemistrv

500 μl of PBMPC-rich elutriation fractions (5 x lOVml.) are placed into the wells of 12

well, sterile multitest slides (ICN) in complete medium and allowed to adhere at 37°C

for 4 hrs. The slides are then placed into 100 x 20 mm. Petri dishes containing 5-7 ml. of DMEM-20%) FCS. The non-adherent cells float off and mesenchymal cells adhere,

spread and grow. Their daily progress is assessed by phase contrast microscopy.

Medium is changed every 3 to 5 days and the cells are studied after 5-7 days. The

growing, adherent cells are rinsed in PBS, fixed in ice-cold 4% paraformaldehyde for

20 minutes and then washed in PBS. All further incubations and washes are carried out

using PBS. Endogenous peroxidase activity is blocked with 0.1 M sodium azide

containing 1% hydrogen peroxide, the specimens are incubated with 10% normal rat

serum, 2%> normal rat serum and 1% bovine serum albumin for 30 minutes at room

temperature to eliminate non-specific binding. Specimens are then incubated with

primary antibodies at 4°C overnight, followed by incubation with a biotinylated

secondary antibody (Vector). The antibody-biotin conjugates are detected with an

avidin-biotin-peroxidase complex (Vector), applied for 30 minutes at room

temperature. A colour reaction develops with 3-amino-9 ethylcarbazole and specimens

are lightly counter-stained with Mayer's haematoxylin. Controls included normal

rabbit or mouse IgG, 1% bovine serum albumin in PBS, or in the case of BMPR

antibodies pre-absorbed with the respective peptide used for immunisation. Quantification of PBMPC bv immunohistochemistrv

5 x 10⁵ PBMPC-rich elutriation fractions in 500 μl of complete medium are placed into

the chambers of sterile, 8 chamber tissue culture treated glass slides (Falcon). Two to 4

hours later non-adherent cells are removed. Cultures are fed every three days. At

regular intervals the slides are rinsed in PBS, fixed in ice-cold 4% paraformaldehyde for

20 min. washed in PBS, stained with anti-BMP receptor antibodies and visualised by

the ABC immunoperoxidase method (described above). The specimens were examined

using an Olympus BH-2 microscope and analysed by computer image analysis

(AnalySIS, Soft Imaging System GmbH, Munster, Germany). Six digital images

(400X) per specimen were recorded and quantitative analysis was performed according

to the colour cell separation. Images, chosen at random were analysed and the data

presented as the mean of the total number of cells per six images examined at 400X.

Slender cells with a small, centrally localised nucleus were scored as fibroblast-like.

Large round cells and intermediate sized cells with more cytoplasm and large round

nucleus were scored as big cells.

Anti-BMPR antibodies

Rabbit polyclonal antibodies to BMP receptors were kindly provided by K. Funa

(Goteborg University). Polyclonal rabbit antisera were prepared using synthetic

peptides corresponding to the intracellular transmembrane portions of the type IA. IB

and II receptors of BMP. The antisera were affinity-purified and tested for specificity by immunoprecipitation of cross-linked complexes of cultured cells transfected with

receptor complementary deoxyribonucleic acids (cDNAs) (10, 11).

Magnetic antibody coated bead separation (MACS , was performed according to the

manufacturer (Miltenyi Biotec, Inc., Auburn, CA.). Elutriation fractions with 50-75%)

monocytes are centrifuged at 900X, washed with MACS buffer (PBS pH 7.2, + 0.5%

SSA + 2mM EDTA) and counted in a hemoacytometer. The cell pellet is resuspended

in 80 μl MACS buffer per 10⁷ total cells. 20 μl of MACS antibody coated beads is

added to the cells, mixed and incubated for 15 minutes at 6-12°C. The cells are washed

with 20X volume of buffer, spun and resuspended in 500 μl buffer. The cell suspension

is applied to a positive selection MS+ column washed previously with 1 ml. MACS

buffer, placed in a magnetic separator and the cells eluted. The column is rinsed four

times with 500 μl buffer and the cells which pass through are combined as the antigen

(-) fraction. The column is removed from the magnetic separator, 1 ml. of buffer is

added to the column and the positive cells flushed out with a syringe plunger. This is

repeated with another 1 ml. of buffer. The elutriated cells are combined as the antigen

(+) fraction.

Alkaline Phosphatase (AP) activity of circulating PBMPC

PBMPC-rich elutriation fractions were prepared from 4 individual blood packs as

described and plated into four well chamber slides (Lab-Tek, Nunc) at 5 x 10⁶ cells per

ml. in DMEM-10%) FCS. After 24 hrs. the non-adherent cells are removed and new medium added containing BMP-2 (a gift from Genetics Institute, Cambridge, MA.,

USA) at concentrations of 0, 1, 10 and 100 ng/ml. The cells are incubated at 37°C in

5% CO₂ and the medium changed every 5 days. Supematants were taken at 5, 10 and

15 days and stored at -20°C for later analysis. AP activity in the supematants is estimated using a p-nitrophenol colorimetric assay (12). Cell supematants are assayed

for AP activity in 50 mM glycine, 0.05% Triton XI 00, 4 raM MgCl₂, and 5 mM

p-nitrophenol phosphate, pH 10.3 for 15 minutes at 37°C (Sigma Diagnostics, St. Louis,

MO). Optical density (OD) is measured at 405 nm and compared to standards.

Stromal Derived Factor (SDF-1) RT-PCR

RNA was isolated from PBMPC cultured for 7-12 days using the Qiagen RN Easy kit (Qiagen, Santa Clarita, CA). RNA is then used for the first strand cDNA synthesis in

the Superscript Preamplification System (GIBCO, BRL. Rockville, MD) according to the manufacturer's instructions. SDF-1 specific primers:

5'-GAGGATCCGACGGGAAGCCC-GTCAGC; 3 '-GAATTCACATCTTGAACCTCTTG.

The annealing temperature is 58°C and the reaction proceeds for 35 cycles. The

glyceraldehyde-3 -phosphate dehydrogenase (GA3PD) gene is included as a RT-PCR

control to normalise for the amount of RNA. Reaction products are analysed in 2% agarose gel containing 0.25 mg/ml. ethidium bromide. RESULTS

PBMPC Selected by Elutriation of Normal Human Blood

When the elutriated cells from the PBMPC fraction were cultured in complete medium

(see methods) without any other supplements they appeared small and round when

examined by phase contrast microscopy. A portion were non-adherent (presumably T

lymphocytes) and were removed with the initial feeding of the culture. After 72 hours

elongated cells with a fibroblast-like morphology and large cells with a clear, thin

adherent cytoplasm around a central nucleus made their appearance. Over the ensuing

7-14 days they became the predominant cells in the culture (Fig. 1A). At higher magnification the fibroblast-like cells often had a splayed, spreading cap at their end

and a small central nucleus. A third population of larger and wider cells with more

cytoplasm and a larger nucleus, were intermediate in their morphology between the large round cells and the thinner fibroblast-like cells (Fig. IB).

Culture conditions modify the morphology of elutriated cells. Adding dexamethasone

(100 nM) at the initiation of the culture significantly reduces (60% + 10%) the total number of cells at the 7th to 10th day and decreases the formation of fibroblast-like

cells (data not shown). Cultures supplemented with a mixture of 100 nM

dexamethasone, plus 0.05 mM ascorbic acid-2-phosphate, and 10 mM β

glycerophosphate (conditions that favour the development of osteoblasts) develop only round or cuboidal cells, but not fibroblast-like cells (Fig. 1C). Dexamethasone alone added at days 6 to 8 reduces fibroblast numbers, but not total cell numbers (data not

shown).

PBMPC-rich elutriation fractions were observed in time-lapse cinematography.

Clusters of small round cells form within 24 hours. Cell processes occasionally extend

from them, but these retract minutes later (Figure 2-left). Individual cells are motile

and often left the field, but the clusters remain intact. After 72 hours, a few cells with a fibroblast-like morphology can be seen beneath and at the edges of the clusters. The

fibroblast-like cells were much larger than the initial cells and quite mobile, extending

and retracting usually about a broad, fixed cup or pseudo-pod. By 6 days, a significant

portion of the cells retain their elongated form and have the large fibroblast-like

appearance of cells in Figure 2-right. Large round cells are also present. Thus, in the

circulation it appears that PBMPC are present as small, round mononuclear cells, but

their subsequent morphology and function is dictated by culture conditions.

Cell Numbers in the PBMPC-rich Elutriation Fraction from 500 ml. of Normal Human Blood

Elutriation fractions were selected for PBMPC quantification based on cell size (intermediate between lymphocytes and monocytes) and granularity (FACS). This

population is comprised of less than 35% lymphocytes and more than 50% monocytes.

Nineteen consecutive samples had an average total cell number of 2.14 + 0.22 (SEM) x

10⁷ of which 64.4% ± 1.5% (SEM) are monocytes. A subpopulation, estimated as 0.3 - 0.7%> of the starting elutriation fractions, was judged to be PBMPC based on their morphology, strong adherence to plastic or glass, and ability to proliferate in

DMEM-20%) FCS without added growth factors (i.e. <1% of the starting 2 x 10⁷ cells in

the elutriation fraction represents 1000 to 10,000 PBMPC). Therefore, in 500 ml. of normal blood there may be several thousand PBMPC.

Cultures were established with 5 x 10⁵ cells from the elutriation fractions and

proliferation measured on days 3, 10 and 17. Non-adherent cells are removed in the

first 24 hours and the cultures fed twice weekly. The adherent cells grow

logarithmically with an approximate doubling time of 2.5 days. By day 17 the initial 5 x 10⁵ cells expand to 6.7 x 10⁷ cells (Table I). The culture conditions are not conducive

to growth of lymphocytes or monocytes, therefore by week 3 mesenchymal cells are the

majority of the proliferating population (<20% CD 14 staining cells-data not shown) in

the cultures.

Cells from an PBMPC-rich elutriation fraction of healthy human blood were cultured in

DMEM-20% FCS. At days 3, 5, 8 and 11 the cultured cells were fixed, stained with

anti-BMPR antibodies and quantified with an autoanalyser (described in Methods).

The results are presented in Figure 3 as the mean of the total number of cells in six

individual images. Big cells (filled bars) plus fibroblast-like cells (hatched bars) are

37%) to 59%o of all the cells, those with a fibroblast-like morphology (hatched bars) vary from 21% to 34% of the PBMPC. The 1 :2 ratio of fibroblast-like to big cells remains relatively stable over 11 days of culture, even as the total cell numbers increase (see Table I). Table I

PROLIFERATION OF PBMPC

OD= optical density see Methods

Imunohistology of Circulating PBMPC

Elutriated cells were grown on sterile 12-well glass slides (ICN) in DMEM-20%o FCS

and 6 to 8 days later the cells are fixed, stained and examined by immunohistology

(Table II). The large cells and the fibroblast-like cells stain with antibodies to both vimentin and collagen type I. They were also identified by antibodies to one or the

other chain of the bone morphogenetic protein receptor (type IA and type II), but did

not react with an anti-type IB receptor chain antibody. Both kinds of PBMPC stain

strongly with anti-CD44 antibody. Conventional T cell (CD3), monocyte (CD 14,

CD68) and B cell (CD20) antibodies stained neither of the two PBMPC populations, nor did they react to anti-LCA (CD45) or MHC-Class II (anti-DR). A monoclonal

antibody, Stro-1, which identifies bone marrow stromal cells, stained most of the large

PBMPC, but only a few of the fibroblast-like cells. Table II

ANTIBODY PROFILE OF PBMPC (DAYS 7-10)

Cell Separation bv Magnetic Beads to Analyse the Contribution of CD34+ Progenitors. Monocvtes or T lymphocytes to PBMPC Formation

PBMPC-rich elutriation fraction were incubated with magnetic beads coated with

specific antibodies and subsequently separated into adherent (antigen enriched) and

non-adherent (antigen depleted) populations. These were cultured in complete medium in 6 well plates (Falcon) and observed daily by phase contrast microscopy. The CD34

depleted fraction always developed many examples of both types of mesenchymal cells

(7 experiments). The fibroblast-like cells appeared in the CD34 depleted cultures at the

same time as in untreated controls (usually day 3 or 4). There were not enough

CD34(+) cells to establish cultures. A representative experiment is shown in Table III.

In two experiments the cells from the elutriation fraction were exposed to CD34 beads

and the negative fraction separated again on fresh CD34 beads. There was no reduction

in the time of appearance or number of fibroblast-like cells. Thus, although PBMPC are present in an elutriation fraction that also contains CD34+ cells, PBMPC and CD34+ cells can be distinguished from one another. Observations with anti-CD 14

beads were somewhat different. Both CD14(+) and CD14(-) eluates develop

fibroblast-like cells in culture, but the numbers were less than in unfractionated

controls. In 4 of 5 experiments the fibroblast-like cells in the CD 14+ fraction appeared

sooner and in greater numbers than the CD 14- fraction. In all instances, however, each

fraction contains both large round cells and fibroblast-like cells and their numbers

become more equal with time (usually by day 13). A representative experiment is

shown in Table III. When the CD14(+) and CD14(-) populations are combined the

number of fibroblast-like and their time of appearance was the same as in unfracti onated controls .

T cell depletion has no effect on the number, morphology or time of appearance of

PBMPC (data not shown).

Table III

MAGNETIC BEAD SEPARATION OF ELUTRIATION FRACTION

* Fibroblast-like cells HPF (Average of 6-10 fields) + = up to 5, ++ = 5-10, +++ = 10-15, ++++ = 15-20 Pleuripotent PBMPC- fibroblast. Osteoblast and Adipocvte Formation

PBMPC-rich elutriation fractions cultured in complete medium support fibroblast

growth. When the same elutriated cells are supplemented with dexamethasone,

ascorbic acid and β glycerophosphate they alter their morphology and become uniform,

polygonal cells reminiscent of osteoblasts (Figure 1C). By 10 days many of the cells

stain for alkaline phosphatase (not shown). Over the next 1-2 weeks a subpopulation

(approximately 30%>) of large (3 to 6X) cells develop, accumulate an ill-defined,

pericellular metachromatic (toluidine blue staining) matrix (not shown), and the osteoblast specific protein - osteocalcin (Figure 4-right). In the same supplemented

cultures are sudanophilic adipocytes (Figure 4-centre) and large cells with multiple nuclei (Figure 4-left). The latter cells stain for tartrate resistant acid phosphatase

(TRAP) (not shown) and the vitronectin receptor, features of osteoclasts (Figure 4-left).

These develop in the supplemented PBMPC cultures because both monocytes and stem cells and/or pre-osteoblasts are present together in the elutriated cells.

PBMPC-rich elutriation fractions from 4 separate blood packs were cultured in

complete medium with varying concentrations of BMP/2 for 5 days and alkaline

phosphatase (AP) in the supematants measured (Figure 5). The lowest concentration of BMP/2 (1 ng/ml.) caused a significant increase in AP activity (p = 0.004). This represents an increased AP production per cell, because BMP-2 had no effect on PBMPC proliferation over 5 days (data not shown). Stromal derived factor ⁽SDF-1 . is a potent CXCα chemokine produced by bone marrow

and other mesenchymal cells, but not blood leukocytes (16, 17) mRNA expression for

SDF-1 was demonstrated in 2 cDNA samples from cultured PBMPCs, and in a cDNA

sample from RA synoviocytes. A plasmid encoding human SDF-1 β was included as a

positive control (Figure 6).

DISCUSSION

The ability to self-replicate and give rise to daughter cells that undergo an irreversible

terminal differentiation are features of stem cells (18). The best characterised are marrow hematopoietic stem cells (MHSC) and their progeny. Friedenstein proposed a

similar scheme for mesenchymal cells and showed that bone marrow contained

primitive cells that could generate progenitors committed to one or another

mesenchymal line (2). Such cells are called mesenchymal stem cells (1). Conditions

that direct marrow mesenchymal stem cells (MMSC) along osteogenic (19, 20),

chondrogenic (19, 21) and stromal pathways (22) have been defined. For instance, in

vitro exposure of fibroblast-like MMSC to optimal concentrations of dexamethasone,

ascorbic acid and β-glycerophosphate induces a cuboidal morphology, upregulates

alkaline phosphatase and osteocalcin, and a mineralised (hydroxyapatite) matrix (20). Lineage differentiation signals can be subtle and vary with the species tested.

Dexamethasone at 10^"9M supports adipocyte differentiation, while at 10^"7M

osteogenesis is favoured (19). Human MMSC obtained by Ficoll density fractionation

(1.078 g/ml.) and cultured in 25%> serum (half horse and half foetal calf) supplemented

with hydrocortisone (1 uM) gives rise to a heterogeneous population and fibroblast-like

cells do not predominate (22). Separation of the same population of MMSC on a Percoll density gradient (1.090 g/ml.) and cultured in carefully selected 10% FCS

resulted in a homogeneous population of spindle-shaped fibroblast-like cells. The

higher density Ficoll may isolate cells that sediment through the Percoll solution used

for the MMSC isolation. Elutriation, as used in this investigation, probably selects a somewhat different population. The CD34 status of PBMPC is disputed (7, 8). A minority of adult BM cells express

CD34. The antigen is present on multipotent HSC, and all unipotent myeloid and

erythroid colony forming cells (23), but CD34 is also recognised on vascular

endothelial cells, basement membrane structures and dermal dendritic and perifollicular

cells in human skin (24, 25). Simmons and Torok-Storb separated human bone marrow

cells based on their CD34 expression (26). More than 95% of the detectable CFU-F

were recovered in the adherent CD34+ population, but their CD34 density was much

less than on CD34^h' HSC. Furthermore, only 5% of the CD34+ marrow cells reacted

with the monoclonal antibody Stro-1, which identifies MMSC (6). All these studies

were done on MMSC prior to culture; after in vitro culture the very same stromal cells

no longer reacted with anti-CD34 antibodies (26). Likewise, although CD34+ cells are

identified in tissue sections of HUVEC, they are not identified in vitro (27). These and

other reports suggest the CD34 glycoprotein is either down-regulated or modified in

vitro to a form that is not reactive with the usual anti-CD34 antibodies (28). Fernandez

failed to demonstrate CD34 on circulating stromal cells mobilised by growth factors,

probably because the cells had been in culture for 10 days (7). Similarly, Majumdar's

inability to demonstrate CD34 staining was on first passage MMSC (22). In vitro

culture conditions, however, cannot explain our failure to eliminate PBMPC in fresh

elutriation fractions with anti-CD34 coated magnetic beads, a technique widely

employed to harvest CD34+ HSC from growth factor mobilised human blood.

Therefore, PBMPC either lack CD34 or have only a very low density of this

glycoprotein. The CD34(+) cells in blood monocyte fractions that develop a fibroblast morphology when grown on fibronectin (fibrocytes) (8) have features identical to

circulating vascular endothelial cell progenitors (28), and are probably not PBMPC.

Bone morphogenetic proteins (BMP) were originally identified as proteins that induced

bone formation at extraskeletal sites (29). Currently, there are twenty or more BMPs,

all members of a larger TGFβ super family. BMPs are involved in morphogenesis and

embryogenesis, influencing bone, cartilage and skeletal formation (12, 29-32). Much of

this information comes from animal cells and embryos, but the addition of BMP-2 to

cultured postnatal human marrow "preosteoblastic" cells in the presence of β

glycerophosphate and ascorbic acid increases the gene message and protein production

of alkaline phosphatase, osteopontin, bone sialoprotein, osteocalcin and alpha- 1

collagen (33). Circulating PBMPC develop into osteocalcin producing cells (Figure

4-right) and make increased amounts of alkaline phosphatase in response to BMP-2

(Figure 5), which is not explained by proliferation, because BMP-2 reduces the number of MMSC in either serum or serum free conditions (33).

BMP receptors (BMPR) belong to the TGF-b receptor family of serine/threonine kinases (34). Both type I and type II BMPR bind their respective ligands, but

heterodimerization is required for a signal to be transduced (11, 34, 35). For instance, coexpression of type II BMPR with either IA or IB BMPR increases ligand binding

affinity and dramatically enhances biologic activity (11). Human MMSC express

BMP-2/4 type I and II receptors as shown in studies employing radiolabelled BMP-2 as ligand in the presence or absence of 100-fold excess of a competitor (33). BMP structure is conserved across species and antibodies to type I and type II receptors react equally well with murine and human mesenchymal cells, but not with hematopoetic

cells (34). This is consistent with our findings that polyclonal antibody to BMP receptors can be used to identify circulating PBMPC and constitutes strong evidence that the circulating cells described in this report are mesenchymal precursors.

Circulating PBMPC stain with the Stro-1 monoclonal antibody made against human

MMSC (6) Stro-1 (+) cells cultured in an osteogenic medium exhibit three markers of

differentiated bone, namely, alkaline phosphatase; 1,25-dihydroxy vitamin D₃

dependent induction of osteocalcin, and a mineralised matrix (hydroxyapatite) (35).

Stro-1 also stains pericytes (36), cells that surround small vessel endothelium.

Pericytes are of mesodermal origin and also have the ability to differentiate into a

variety of different cell types, including osteoblasts and adipocytes (reviewed in 37).

However, BMPR antibodies have been used to analyse mesenchymal precursor cells in synovial tissues and while they stain large cells in the inflamed joint lining no staining is observed in blood vessels of normal or inflamed synovium (L. Marinova -

Mutachiefa submitted for publication).

Progenitor and precursor B cells require close contact with MMSC for growth and

maturation. Mouse MMSC contain the gene for a protein (termed either stromal cell-derived factor 1 (SDF-1) or pre-B cell growth stimulating factor-(PBSF) (16, 17)

SDF-1, a powerful CXC chemokine, that recruits circulating lymphocytes, monocytes

and CD34(+) hematopoietic progenitors, but not neutrophils (39, 40). PBSF is

responsible for converting "early" B cells into immunoglobulin producing cells (41), SDF-1 mRNA is constitutively expressed in many tissues, unlike other chemokines, which are only induced (41, 42). SDF-1 is expressed in MMSC, dermal f broblasts and synovial fibroblasts, but not HSC. The demonstration of constitutive expression of

SDF-1 mRNA in cultured circulating PBMPC (Figure 6) and in supematants from

cultured circulating PBMPC (data not presented) is additional evidence that PBMPC

belong to a mesenchymal lineage. Human osteoclasts (OC) arise from HSC and/or

blood monocytes in close proximity to stromal cells (15). The osteoclast is a TRAP

positive, large multinucleated cell with receptors for calcitonin and vitronectin (αvβ3)

(see Figure 4-left) and the capacity to form resorption lacunae in bone slices.

Osteoblast and OC production is tightly linked and regulated. Osteoblasts facilitate OC formation by providing physical support and critica' soluble factors (43). Our

observations of the spontaneous formation of cells with the morphology and phenotype

of OC in a monocyte rich (65%) elutriation fraction is best explained by the

simultaneous presence of PBMPC in the same fractions.

Every one of more than 100 subjects had cells in an appropriate fraction of elutriated blood cell that fulfil criteria for mesenchymal precursors or stem cells. They proliferate

rapidly in culture with an adherent spread morphology, display cytoskeletal,

cytoplasmic and surface markers of mesenchymal progenitors and possess a capacity

for differentiation into fibroblast, osteoblast, and adipocyte lineages. Thus, autologous

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Claims

1. An isolated periferal blood-derived mesenchymal precursor cell (PBMPC).

2. An isolated PBMPC obtainable by elutriating human blood.

3. An isolated PBMPC according to claim 2, obtained by the steps of:

(i) obtaining a sample of human blood;

(ii) depleting the sample of blood of red blood cells;

(iii) elutriating the red blood cell-depleted sample; and

(iv) selecting one or more aliquot(s) of elutriated sample containing elevated

PBMPC concentration.

4. An isolated PBMPC according to claim 3, wherein the selected aliquots (iv) contain between 50%) and 75%> monocytes of the total number of cells within the aliquot.

5. An isolated PBMPC according to claim 3 or claim 4, wherein the sample of

blood is a sample of anti-coagulated platelet-depleted leucocytes.

6. An isolated PBMPC, wherein the selected aliquot(s) are then (v) centrifuged, washed, plated in growth medium and allowed to grow.

7. Use of an PBMPC according to any preceding claim in tissue engineering or gene therapy.

8. A method of producing a differentiated cell comprising culturing an isolated

PBMPC according to any one of claims 1 to 6 under cell tissue culture conditions to produce differentiated cells or tissue.

9. A method of isolating an PBMPC comprising the steps of:

(i) obtaining a sample of human blood;

(ii) elutriating the sample of human blood; and

(iii) selecting one or more aliquots of elutriated sample containing enriched PBMPC numbers.