STROMAL STEM CELLS

Abstract

Stromal stem cells are prospectively isolated from human bone marrow then expanded into clonal populations and cultured and used, the isolation being on the basis of expression of a cell surface marker, wherein the cell surface marker binds an antibody and wherein said antibody cross reacts with a cell surface marker found on mouse stromal stem cells or rat stromal stem cells, and optionally also on a cell of at least one other mammalian species selected from mouse, rat, horse, rabbit and pig cells. Useful stromal stem cell populations are positive for SDC2.

Claims

1. A composition comprising: (i) a population of mammalian stromal stem cells, wherein 30% or more of the cells are positive for SDC2; and (ii) a cryopreservant, wherein the population of mammalian stromal stem cells exhibits at least 10-fold more colony forming units per 10{circumflex over ( )}5 cells plated compared with native pre-sorted mononuclear cells.

2. The composition of claim 1, wherein the cryopreservant is dimethylsulfoxide, human serum albumin, or hyaluronic acid.

3. The composition of claim 1, further comprising saline or collagen.

4. The composition of claim 1, wherein the cells are derived from bone marrow, umbilical cord, adipose tissue, skeletal muscle, endometrium, placenta, umbilical cord blood, or Wharton's jelly.

5. The composition of claim 1, wherein 30% or more of the cells are pre-chondroblast cells.

6. The composition of claim 1, wherein 75% or more of the cells are positive for SDC2.

7. The composition of claim 1, wherein 75% or more of the cells are osteo-lineage precursor cells cells.

8. The composition of claim 1, wherein less than 25% of the cells are pre-adipocyte cells.

9. The composition of claim 1, wherein the cells are human cells.

10. The composition of claim 1, wherein the cells are negative for CD45.

11. The composition of claim 1, wherein the composition is suitable for injection.

Description

[0085] The invention is now described in specific embodiments with reference to the accompanying drawings in which:

[0086] FIG. 1 shows labelling by anti-SDC2 antibody of human stromal stem cells but not human lung fibroblasts. Green lines indicate anti-SDC2− APC staining of cells. Red lines indicate labelling with appropriate control antibody (Rat lgG2B Isotype Control-APC; R&D #IC013A; Clone −141945). Blue lines indicate positive control labelling of MRCS with anti-PDGFRa-APC antibody (R&D Systems #FAB1264A; Clone—PRa292);

[0087] FIG. 2 shows labelling by SDC2-APC antibody of CD271.sup.bright/.sup.DC45low human bone marrow mononuclear cells. Data show fluorescence-activated Cell Sorting (FACS) profile of 3.5 ×10.sup.7 BMMNCs stained with aforementioned SDC2-APC, CD271-PE and CD45-FITC (both from BD). CD45-FITC staining permitted gating of BMMNCs into 3 populations, (A) CD45-ve—BLUE, (B) CD45low—ORANGE, and (C) CD45high—GREEN. In (B) rare TP SDC+ve/CD271 bright/CD45low cells are BLUE;

[0088] FIG. 3 shows enhanced enrichment in CFU-F in SDC+/CD271.sup.bright/DC45.sup.low sorted bone marrow mononuclear cells. Data show fluorescence-activated Cell Sorting (FACS) profile 10.sup.7 BMMNCs stained with aforementioned SDC2-APC, CD271-PE and CD45-FITC (both from BD);

[0089] FIG. 4 shows percentage of wells of a 96 well plate in which no clone formed as function of number of SDC+/CD271.sup.bright/CD45− mononuclear cells per cell;

[0090] FIG. 5 shows the number of population doublings of clones;

[0091] FIG. 6 shows in vitro GAG deposition of SDC2+ stromal stem cells at 2% and 19% oxygen tension. Representative Safranin-O stained histology sections from SDC2+SSC derived micromass pellets are shown;

[0092] FIG. 7 shows in vitro lipid deposition of SDC2+ and SDC2− (labelled as S2+ and S2− respectively) stromal stem cells;

[0093] FIG. 8 shows calcium deposition of SDC2+ and SDC2− stromal stem cells in response to in vitro osteogenic stimuli;

[0094] FIG. 9 shows GAG deposition of SDC2+ and SDC− stromal stem cells in response to in vitro chondrogenic stimulation;

[0095] FIG. 10 shows relative HUVEC cord formation of SDC2+ and SDC2− stromal stem cells in response to in vitro angiogenic stimulation;

[0096] FIG. 11 shows that rare CD45−/SDC+ human bone marrow mononuclear cells express stromal stem cell marker CD271, CD146 and NG2;

[0097] FIG. 12 shows SDC2 expression of stromal stem cells isolated from and rabbit bone marrow;

[0098] FIG. 13 shows increasing levels of SDC2 in stromal stem cells derived from equine bone marrow upon confluence, and stromal stem cells derived from pig bone marrow express SDC2 in low oxygen tension;

[0099] FIG. 14 shows increasing levels of SDC2 in stromal stem cells derived three strains of rat bone marrow upon confluence;

[0100] FIG. 15 shows FACS isolation of CD45− mononuclear cell with co-stain for SDC2 and Scal; and

[0101] FIG. 16 shows enhanced enrichment in CFU-F in SDC+/Scal selected mouse mononuclear cells.

[0102] We used a rat lgG2B monoclonal antibody to human SDC2 conjugated to the Allophycocyanin (APC) fluorochrome (R&D Systems number FAB2965A; clone 305515) to indicate if expression of SDC2 protein is enriched on the cell surface of human SSC in comparison to a control human foetal lung fibroblast cell line, MRCS. MRCS lung fibroblasts cultured in SSC growth media (aMEM/10% PAA FSC; NUNC T175 flasks) did not express SDC2 (FIG. 1). As a control, we show that MRCS fibroblasts do express the PDGFRa marker (CD140a-APC). These data suggests that expression of SDC2 protein is enriched on the surface of human SSC in comparison to control lung fibroblasts (FIG. 1).

[0103] At this time, the state of the art for antibody-based purification of SSC from BM consists of using a combination of anti-CD271 (LNGFR) and anti-CD45 antibodies, reported by University of Leeds (Drs. McGonagle and Jones). This isolation of CD45low/CD271 bright cells has been shown to capture all CFU-F (SSC).

[0104] However, the definition of CD271 ‘bright’ cells can be difficult to standardise from lab to lab. To investigate if this anti-SDC2 antibody co-stains CD45low/CD271 bright BMMNCs, 30 ml of human BM was aspirated from donor CRFG #0007 at the Clinical Research Facility (CRF) at Galway University Hospital (GUH) by Dr Ruth Morrell.

[0105] BMMNCs (5 ×10.sup.8) were isolated by Ficoll centrifugation, washed in PBS, resuspended in MACS buffer and blocked with Human FC-Block (Miltenyi, UK). BMMNCs (4 ×10.sup.7) were stained with anti-SDC2-APC (R&D), anti-CD271-PE (BD) anti-CD45-FITC (BD) and Sytox/DAPI viability dye. Cells were analysed by FACS on the Becton Dickinson Ariall at NUI Galway.

[0106] FIG. 2 indicates representative histogram/dot plots from SDC2/CD271/CD45 triple stained cytometry experiments. FIG. 2B indicates BMMNCs that express low/mid levels of the CD45 marker (orange). In agreement with other reports, we find that CD271-positive cells are found within the CD45low population (FIG. 2B) and in these experiments, we noted that the anti-SDC2-APC antibody labelled CD45low/CD271-positive cells. Specifically, the anti-SDC2-APC antibody labels CD45low/CD271 bright BMMNCs. The SDC2+/CD45low/CD271+ triple positive (TP) population are rare within BMMNCs with a frequency ranging from 1:16,000 to 1:23,000.

[0107] FIG. 3 shows that SDC2+/CD271+/CD45− MNC fraction contain 3000-fold more CFU-F/S SC compared to native pre-sorted BMMNCs. Conversely the SDC2-negative fraction of the CD271+ population does not retain a significant number of CFU-F/SSC

[0108] Single cell FACS sorting experiments were performed to enumerate the number of clonogenic cells within the SDC2+/CD271+ population. Single SDC2+/CD271+/CD45− MNC were sorted at 1, 3 and 20 cells per well in a 96 well plate. A limiting dilution analysis reveals that, at lcell per well, 16-17 clones formed per 96 well plate (FIG. 4). All 16 clones were proliferative and able to undergo 15-20 population doublings (FIG. 5). Notably, selected SDC2+ clones were able to undergo significant chondrogenesis in response to in vitro micromass culture. All five clones exhibited enhanced glycosaminoglycan (GAG) deposition when cultured in low (2%) tensions of oxygen (FIG. 6).

[0109] When compared to pre-sorted (parental) SSC, FACS-sorted and culture expanded SDC2+SSG exhibit significantly attenuated deposition of lipids in response to in vitro stimulation with potent adipogenic media over a 14 day differentiation regimen (FIG. 7), as visualised with Oil Red O staining, extractions and quantification. Conversely, compared to SDC2-SSC and pre-sort SSC, SDC2+ SSC elicit enhanced deposition of calcium and enhanced matrix mineralisation in response to a 14 day induction with an osteogenic media, as measured by calcium extraction and Alizarin Red S staining respectively (FIG. 8). Notably, no difference was observed between the three populations of SSC when subjected to chondrogenic micromass culture (FIG. 9).

[0110] Human vascular endothelial cells (HUVEC) can form angiogenic cord-like tubules within 24 hours of being plated on nutrient-rich matrigel. Co-culture of SDC2+ SSC at a ratio of 1:1 with HUVEC on matrigel elicits a 3-fold increase in the number of stable vascular tubules at 24 hours (FIG. 10).

[0111] Finally, Human SDC2+/CD45− MNC also express key stromal markers including CD146, NG2 (CSPG4) and CD271 (FIG. 11).

[0112] FIG. 12 represents flow cytometry histograms of SSC derived from BM and Adipose MNC of goat and rabbit BM tissue. While the SDC2 marker does not appear to be detectable on goat SSC, significant levels of SDC2 protein can be detected on rabbit SSC (FIG. 12).

[0113] An increased detection of SDC2 protein is increased in cultured equine SSC in response to confluent culture (FIG. 13). SDC2 protein can also be detected in a sub-population of porcine SSC when cultured in low oxygen tension (FIG. 13).

[0114] SDC2 marker is expressed on the surface of rat SSC (FIG. 14). As seen in equine SSC, SDC2 protein increases in the surface of rat SSC in response to confluence. This pattern can be seen in SSC derived from the marrow of three typically used laboratory strains of rat, namely, Dark Agouti, Sprague Dawley and Lewis (FIG. 14).

EXAMPLES

Example 1.1—Isolation of Bone Marrow Aspirates

[0115] Human bone marrow samples were obtained from the posterior iliac crest of healthy volunteers (n=3) following written consent from the patients. Patients underwent virology testing for HIV I and II, Hep A/C, HBsAg, Anti-HB core, Syphilis and CMV in accordance with EU Tissue Directive 2006/17/EC requirements. In a BSC, samples are pooled and divided into 7.5 mL aliquots and subjected to density-gradient centrifugation.

Example 1.2—Isolation and Expansion of Human SSC by Density-Gradient Centrifugation (Ficoll)

[0116] In a biological safety cabinet under aseptic techniques, 7.5 mL of Ficoll is pipetted into 50 ml centrifuge tubes. To remove clots, the 30 ml of BM was filtered through a 100 micron cell sieve (BD Falcon) into a 50 ml centrifuge tube. Filtered marrow was diluted 1/1 in D-PBS and then split evenly between the 4 tubes containing Ficoll, slowly pipetting the BM onto the side of the tube lying at an angle of 35° to 45° to insure a slow release of BM, without disturbing Ficoll or producing bubbles. Tubes were then centrifuges for 22 mins at 900 g with centrifuge brakes set to zero to form a fractionated sample. After centrifugation, tube contents formed three layers; a top layer of plasma, a thin layer—Buffy coat—contains the MNC, a clear layer of Ficoll and a bottom layer containing red blood cells constituents—erythrocytes and granulocytes. The Buffy coat was carefully aspirated out being careful not to disturb the surrounding cells. These cells were then transferred to another 50 mL centrifuge tube and resuspended in 45 mls D-PBS. These tubes were then centrifuged for 10 mins at 350 g. Supernatant was aspirated and pellets resuspended in 5 mL complete media. These were then centrifuged for 10 mins at 350 g. Supernatant was aspirated and pellets were pooled in 5 mL D-PBS.

Example 1.3—CFU-F Plates Seeding

[0117] After isolation of mononuclear cells via direct plating or Ficoll, 9 ×10.sup.6 cells were isolated from both sets of cells and seeded in 10 cm dishes in triplicate at a seeding density of 3 ×10.sup.6 MNC/plate. These cells were washed and fed at same time as cells in culture as outlined above.

Example 1.4—Crystal Violet CFU-F Staining

[0118] On days 12-14, cells were fixed and stained for CFU-F analysis. Media was aspirated from plates and plates were washed three times in D-PBS to remove and remaining media. Cells were fixed by pipetting 8 mL 95% Methanol, stored at −20° C., onto cells for 10 mins and gently swirling. Methanol was aspirated from plates and cells were washed once with D-PBS. 8 mL of crystal violet (0.5% crystal violet, 99.5% Methanol) was then added to plates and plates were gently swirled. Plates were left for 10-15 mins. Excess crystal violet was aspirated and cells were washed three times with D-PBS to remove and remaining excess stain. Plates were then inverted and left to dry overnight. Dry plates were then imaged using a flat bed scanner. Colonies were then counted and characterised by visual inspection under an inverted light microscope (Olympus CKx41). Colonies comprised of clusters greater than 50+ were counted as a CFU.

Example 2—Antibody Analysis of Mononuclear Cells and Stromal Stem Cells

[0119] Table 1 shows the details of the antibodies used to profile the mononuclear cells and stromal stem cells produced in Example 1.

TABLE-US-00001 TABLE 1 Antibody Supplier Catalogue No. CD362/Sydecan-2 R&D Systems N/A CD271/LNGFR BD N/A W8B2/TNAP/ALP Miltenyi N/A TWEAK/TNFSF13 BD N/A APRIL/CD256 BD N/A CD146 BD N/A CD105 Invitrogen MHCD10504 CD73 BD 550257 CD90 BD 555596 CD14 AbD Serotec SPL2185 CD19 BD 345777 CD3 BD 345765 CD34 BD 555822 CD45 BD 555483 ┌1, γ2a controls BD 342409 HLA-DR Invitrogon MHLDR04 ┌2b control Caltag MG2b04

Blocking Solution Preparation

[0120] Blocking solution was prepared by adding 1 mL of FBS to 49 mL of D-PBS in a 50 mL tube.

Sample Preparation

[0121] Cells were trypsinised at 37° C. and transferred to culture media in 15 mL tube. Cells were centrifuged for 5 mins at 400 g. Supernatant was aspirated and cells resuspended in 5 mL complete culture media. Cell counts and viability testing were performed using Trypan blue. Cells were then centrifuged for 5 mins at 400 g and supernatant aspirated. Blocking solution was then added to cell pellets to resuspend cells at 1 ×10.sup.6 cells/mL.

Staining of SSC (Analysis on FACS Canto)

[0122] PE-labelled antibodies were removed from refrigeration and placed on ice along with a 96 well round bottomed plate (Sarstedt) and blocking solution. 1 ×10.sup.5 cells (100 μL) was pipetted into each of 12 wells of the 96 well plate on ice, one for each antibody and 1 for unstained cells. Plate was then centrifuged for 4 mins at 400 g, 4° C. Supernatant was aspirated carefully to not disturb cell pellet and 50 μL of blocking solution was added to each well and pellet resuspended by pipetting of solution.

Example 3—Chondrogenic Differentiation of SSC

[0123] Table 2 shows the composition of the incomplete chondrogenic media (ICM).

TABLE-US-00002 TABLE 2 Reagent Volume Final Concentration DMEM (HG) 95 mL Dexamethasone 1 mM 10 μL  100 nM Ascorbic acid 2-P: 5 mg/mL  1 mL   50 μg/mL L-Proline: 4 mg/mL  1 mL   40 μg/mL ITS + supplement  1 mL 6.25 μg/mL bovine insulin 6.25 μg/mL transferrin 6.25 μg/mL selenous acid 5.33 μg/mL linoleic acid 1.25 mgmL BSA Sodium pyruvate  1 mL   1 mM Penicillin/Streptomycin  1 mL  100 U/mL penicillin  100 μg/mL streptomycin

[0124] Cells were thawed using 37° C. water bath and quickly transferred to culture media in 15 mL tube, washing out the cryovial with 1 mL of media. Cells were centrifuged for 5 mins at 400 g. Supernatant was aspirated and cells resuspended in 5 mL complete culture media. Cell count was performed and enough cells were harvested to form pellets of between 2-2.5 ×10.sup.5 cells/pellet. 4 positive cultures and 2 negative cultures were set up for each sample. Cells were centrifuged again for 5 mins at 400 g to remove culture media. Supernatant was aspirated and cells resuspended in 3 mL incomplete chondrogenic media (ICM). 3 mL cell suspension was divided into 15 mL tubes (2 mL for positive pellets, 1 mL for negative pellets). Tubes were centrifuged for 5 mins at 100 g. Cells for positive pellets were resuspended in 500 pL of complete chondrogenic media (CCM) for every pellet to be formed. CCM consists of ICM with 0.5 μL of TGF-β per mL of ICM.

[0125] Cells for negative pellet were resuspended in 500 μL of ICM for every pellet to be formed. Cells were transferred to screw cap Eppendorf tubes and centrifuged for 5 mins at 100 g in a swing out rotor. Tube caps loosened to allow gas exchange and incubated in BSC at 37° C., 5% CO.sub.2. Media was changed every second day by aspirating as much of the media as possible without disturbing the pellet and replacing with either CCM or ICM for positive pellets and negative pellets respectively. At day 21, cell pellets were harvested by aspirating off all the media and washing twice in D-PBS. Pellets were allowed to air dry and 3 of the 4 positive pellets were used for GAG measurement and the other one was used for histology. Pellets for GAG measurement were stored at −20° C. and pellet used for histology was fixed in 10% formalin for 1 hr and then stored in water until ready to be processed.

Example 4—Chondrogenic Assay

Preparation of DMMB Stock Solution

[0126] 16 mg of DMMB was dissolved overnight in 5 mL of reagent grade 100% ethanol. 2.73 g NaCl and 3.04 g Glycine was added to 975 mL of deionised water. 0.69 mL of conc HCl (11.6M) was added to this solution and mixed. Dissolved DMMB was added to this solution. Container of DMMB was then rinsed repeatedly with Dl water until all of DMMB solution was transferred. pH was adjusted to 3.0 with 1 M HCl. Volume was adjusted to 1 L with deionised water and solution was protected from light by wrapping in tinfoil.

[0127] Papain solution was prepared by dissolving 1 mg of papain (Sigma P4762) in 9.75 mL of warm diluted buffer. Diluted papain was prepared by adding 250 μL of this solution to 10 mL of dilution buffer.

[0128] 200 μL of papain solution was added to each pellet and allowed to digest overnight in 60° C. oven. Samples were then vortexed to disperse pellet. Standards were made up using chondroitin-6-sulphate (Sigma C4384) by adding 4 mg of chondroitin-6-sulphate to 10 mL of dilution buffer making a 400 m/mL stock. This was then diluted to give a 40 μg/mL solution. Dilutions were made as follows from this 40 μg/mL solution as shown in table 3.

TABLE-US-00003 TABLE 3 Chondroitin sulphate Concentration solution Dilution GAG/well (40 μg/mL) Buffer (50 μL) 200 μL  0 μL   2 μg 180 μL  20 μL 1.8 μg 160 μL  40 μL 1.6 μg 120 μL  80 μL 1.2 μg  80 μL 120 μL 0.8 μg  40 μL 160 μL 0.4 μg  0 μL 200 μL   0 μg

[0129] 50 μL of standards and samples were added in triplicate to each well of a 96 well plate. 200 μL of DMMB stock solution was added to each well and incubated at room temperature (RT) for 5 mins. Plates were read using an absorbance plate reader at 595 nm. Absorbance readings for standards containing 0 μL GAG/well where used as a blank value and subtracted from other absorbance readings.

Measurement of DNA Using PicoGreen

[0130] 1×TE solution was prepared by diluting the 20×stock solution provided in the Quant-iT Kit (Sigma P7589) 1 in 20 parts in distilled water. A diluted PicoGreen solution was prepared by diluting DMSO to 1 in 200 parts dH.sub.20. DNA stock (100 g/mL) was diluted in 1×TE 50-fold to give a final concentration of 2 g/mL DNA standards were prepared as shown in table 4.

TABLE-US-00004 TABLE 4 DNA Working Final conc Stock 1 × TE DNA/mL 400 μL 0 2000 ng 200 μL 200 μL 1000 ng 100 μL 300 μL  500 ng  40 μL 360 μL  200 ng  20 μL 380 μL  100 ng  10 μL 390 μL  50 ng  4 μL 396 μL  10 ng  0 μL 400 μL   0 ng

[0131] Papain-digested samples (outlined above) were further diluted 25-fold in 1×TE. 100 pL of standards and samples were added in triplicate to a 96-well black plate. Plate must be black as reaction is affected by light. 100 pL of PicoGreen solution was added to each standard and sample well and allowed to incubate for 2-3 mins. Plates read on fluorescent plate reader by first exciting plate at 485 nm and then reading plate at 538 nm.

Example 5—Adipogenic Differentiation of SSC

[0132] Table 5 shows the composition of the adipogenic induction media.

TABLE-US-00005 TABLE 5 Volume (to make Reagent 100 mL) Final Concentration DMEM (HG) 87.6 mL  1 μM Dexamethasone 1 mM  100 μL Insulin 1 mg/mL   1 mL  10 μg/ml Indomethacin 100 mM  200 μL 200 μM 500 mM MIX  100 μL 500 μM Penicillin/Streptomycin   1 mL 100 U/mL penicillin 100 μg/mL streptomycin FBS   10 mL 10%

[0133] Table 6 shows the composition of the adipogenic maintenance media.

TABLE-US-00006 TABLE 6 Volume (to make Reagent 100 mL) Final Concentration DMEM (HG) 88 mL Insulin 1 mg/mL  1 mL  10 μg/ml Penicillin/Streptomycin  1 mL 100 U/mL penicillin 100 μg/mL streptomycin FBS 10 mL 10%

[0134] Cells were thawed using 37° C. water bath and quickly transferred to culture media in 15 mL tube, washing out the cryovial with 1 mL of media. Cells were centrifuged for 5 mins at 400 g. Supernatant was aspirated and cells resuspended in 5 mL complete culture media. Cell count was performed and enough cells were harvested to seed cells at confluency (4 ×10.sup.4 cells/well) in a 24 well plate with flat bottom. 4 test wells and 4 control wells were set up. Cells were seeded in 1 mL of culture media in each well. Cells were incubated at 37° C., 5% CO.sub.2 and after 48 hrs cells were viewed to have adhered to the plastic and appeared confluent. To test wells, complete culture media was replaced with 1 mL of adipogenic induction media and left for 3 days. Control wells were replaced with complete culture media. After 3 days in adipogenic culture media, media in test wells was replaced with 1 mL/well of maintenance media and left for between 1 and 3 days. This was then replaced with 1 mL/well of maintenance media. This process was repeated three times. After the final media change to maintenance media, cells were left in media for 5 to 7 days before harvesting.

Example 6—Adipogenic Assay Oil Red O Staining

[0135] A working solution of Oil Red O was prepared by mixing 6 parts of Oil Red O stock solution with 4 parts of dH.sub.2O. Solution was allowed to stand for 10 mins and then filtered through Whatman no. 1 Filter paper.

[0136] Media was aspirated and cells washed twice in D-PBS. Cells were then fixed in 10% formalin for 1 hr at RT. Formalin was aspirated and plates rinsed in dH.sub.2O. 500 pL of Oil Red O working solution was pipetted to each well to cover layer of cells. Plate rotated slowly in FIG. 8 motion to spread Oil Red O over cells evenly and left for 5 mins. Stain was aspirated and excess stain was removed by adding 2 mL/well of 60% Isopropanol. Plates were again swirled in FIG. 8 motion and Isopropanol aspirated. Plates rinsed with tap water until water ran off plate smoothly. Samples were then stored in dH.sub.20 until imaging.

Extraction of Stained Lipid

[0137] After imaging of samples, water was aspirated from wells. Oil Red O was extracted by pipetting Isopropanol (2 ×500 pL) over the surface of the wells several times. Isopropanol and dye were then transferred to an Eppendorf tube. Samples were centrifuged for 2 mins at 500 g to pellet and debris in samples. 200 μL of the extracted stain for each sample was added in triplicate to a 96 well plate. Staining was measured using a plate reader at 520 nm.

Example 7—Osteogenic Differentiation of SSC

[0138] Table 7 shows the composition of the osteogenic differentiation media.

TABLE-US-00007 TABLE 7 Volume (to make Reagent 100 mL) Final Concentration DMEM (LG) 87.5 mL Dexamethasone 1 mM   10 μL 100 nM Ascorbic acid 2-P 10 mM   1 mL 100 μM B glycerophosphate   1 mL  10 mM FBS   10 mL 10% Penicillin/Streptomycin   1 mL 100 U/mL penicillin 100 μg/mL streptomycin

[0139] Cells were thawed using 37° C. water bath and quickly transferred to culture media in 15 mL tube, washing out the cryovial with 1 mL of media. Cells were centrifuged for 5 mins at 400 g. Supernatant was aspirated and cells resuspended in 5 mL complete culture media. Cell count was performed and enough cells were harvested to seed cells at confluency (4 ×10.sup.4 cells/well) in a 24 well plate with flat bottom. 4 test wells and 4 control wells were set up. Cells were seeded in 1 mL of culture media in each well. Cells were incubated at 37° C., 5% CO.sub.2 and after 48 hrs cells were viewed to have adhered to the plastic and appeared confluent. Media in test wells was replaced with osteogenic media and media in control wells was replaced with complete culture media. Media in all wells was changed twice weekly. Cells were harvested between days 10 and 17.

Osteogenic Assay

[0140] 1 of 4 test wells and control wells are used for Alizarin Red staining. The other 3 were used for calcium quantification.

Alizarin Red Staining

[0141] 2% Alizarin Red S solution was prepared by dissolving 2 g Alizarin Red S in 100 mL dH.sub.2O. Solution was mixed and pH was adjusted to approximately 4.1-4.3 using 1% ammonium hydroxide as pH is essential for staining process. Media was aspirated from wells. Cells were washed twice in D-PBS to remove remaining media to insure no staining of media occurred. 95% methanol was prepared by diluting 95 mL 100% methanol with 5 mL water. Methanol was then stored in ice to low temperature. Cells were fixed in ice cold Methanol for 10 mins. Methanol was aspirated and cells were rinsed in dH.sub.2O. 500 pL of 2% Alizarin Red S was added to wells and left for 5 mins, occasionally gently swirling the plate in FIG. 8 motion. After 5 mins calcium staining was visible. Cells rinsed in dH.sub.2O and imaged using an Olympus CKx41.

Calcium Assay

[0142] 0.5M HCl was prepared by diluting 4.3 mL 11.6M HCl in 95.7 mL water. Media was aspirated from wells and wells washed twice in D-PBS to remove any remaining media. 0.2 mL 0.5M HCl was added to each well and cells were scraped from wells using a cell scraper and collected in labelled Eppendorf tubes. Solution was left shaking overnight on cell shaker in a dark cold room. Samples centrifuged briefly to pellet cell debris. Calcium assay was performed using a Stanbio Kit. A working solution of 1:1 of binding reagent and working dye were prepared.

[0143] Table 8 shows the composition of calcium assay standards.

TABLE-US-00008 TABLE 8 Volume Concentration 10 mg/dl (μg/well) std/well 0 0 0.05 0.5 μL 0.10   1 μL 0.2   2 μL 0.4   4 μL 0.6   6 μL 0.8   8 μL 1.0  10 μL

[0144] Standards and samples were plated in triplicate in a 96 well plate. 10 μL of 0.5M HCl was added to each standard well. 10 pL of samples were added to each well. 200 μL of working solution was added to every standard and sample well. Absorbance was read at 550-650 nm using a Victor3™ 1420.

[0145] SDC2 co-stained with Scal on the surface on CD45-ve murine BMMNC from the C57/B16 strain. Moreover, FACS sorting of SDC2+/Sca1+ MNC from murine marrow reveals that SDC2 marks a self-renewing sub-population of SSC that can form CFU-F at significantly increased frequencies compared to plated pre-sorted MNC.

Example 8—SDC2.SUP.+ Cells from Human Pluripotent Cells

[0146] We obtained populations of cells expressing SDC2 from human pluripotent cells, in this case ES cells (ES−), for comparison with cells derived from bone marrow (BM−).

[0147] BM-SSCs and ES-SSCs (Millipore Human Mesenchymal Stem Cells (derived from hES cells)) were plated at a density of 10.sup.5 cells per well of a 6-well plate (Nunc) in complete media (BM-SSCs: α-MEM, 10% FBS; ES-SSCs: Millipore FibroGRO™ LS Complete Media Kit) and left to adhere overnight. Cells were harvested when they reached subconfluent levels (˜60% confluent), and confluent levels (100% confluent).

[0148] The results from flow cytometric analysis of “classical” SSC markers illustrated that BM-SSCs and ES-SSCs had similar expression of CD73. The expression of the marker CD105 remained the same for both confluent and sub-confluent cultures; CD105 expression appeared to decrease with increasing confluency. The expression of SDC2 by BM- and ES-SSCs remained consistent in confluent and sub-confluent culture conditions; the percentage population BM-SSCs expressing SDC2 increases in confluent culture and is consistently high for ES-SSCs in both confluent and nonconfluent cultures. The RFI of SDC2 expression by ES-SSC is higher.

[0149] Hence, hES derived stromal stem cells expressed SDC2 and therefore cell populations enriched for SDC2 can be obtained direct from human pluripotent cells including hES and hiPS cells.

Example 9—SDC2* Cells in Treatment of Ventilator Induced Lung Injury in Rats Methods and Materials

[0150] All work was approved by the Animal Ethics Committee of the National University of Ireland, Galway and conducted under license from the Department of Health, Ireland.

hSSC Isolation and Culture

[0151] Human SSCs (hSSCs) were isolated from adult volunteers as previously described. Following aspiration, the bone marrow was plated into tissue culture flasks. Adherent cells were grown until 80% confluent and then trypsinized and culture expanded to passage 4, whereupon they were used for experiments. SSCs were characterized according to international guidelines. Fibroblasts, used as control cells, were obtained from a stable cell line as previously described.

Series 1 [Ventilation Induced Lung Injury]

[0152] Adult male Sprague Dawley rats were anaesthetised, orotracheally intubated and randomized to undergo injurious mechanical ventilation. [0153] The following ventilator settings were used: P.sub.Insp 35 cmH.sub.2O, respiratory rate 18 min.sup.−1, and PEEP 0 cmH.sub.20. When respiratory static compliance had decreased by 50% the animals were allowed to recover. [0154] Following recovery, animals were randomized to intravenous administration of: (i) vehicle (PBS, 300 μL); (ii) fibroblasts (4 ×10.sup.6 cells); (iii) human SSCs (4 ×10.sup.6 cells) or (iv) cells of the invention, referred to as human S2.sup.+SSCs (4 ×10.sup.6 cells); in a four group design. [0155] The extent of recovery following ALI and the inflammatory response was assessed after 24 hours.

Series 2 [Low Stretch ‘Protective Ventilation]

[0156] Adult male Sprague Dawley rats were anaesthetised, orotracheally intubated and randomized to low stretch mechanical ventilation. [0157] The ‘low stretch’ protocol comprised of mechanical ventilation for 90 minutes with the following settings: FiO.sub.2 of 0.3, respiratory rate 80.min.sup.−1, tidal volume 6 ml.Math.kg.sup.−1 and positive end-expiratory pressure of 2 cm H.sub.2O [0158] Following recovery, animals were randomized to intravenous administration of: (i) vehicle (PBS, 300 μL); (ii) fibroblasts (4 ×10.sup.6 cells); or (iii) intra-tracheal human SSCs (4 ×10.sup.6 cells); in a six group design. [0159] The extent of recovery following ALI and the inflammatory response was assessed after 24 hours.

Statistical Analysis

[0160] The distribution of all data was tested for normality using Kolmogorov-Smirnov tests. Data were analyzed by one-way ANOVA, followed by Student-Newman-Keuls, or by Kruskalis-Wallis followed by Mann-Whitney U test with the Bonferroni correction for multiple comparisons, as appropriate. Underlying model assumptions were deemed appropriate on the basis of suitable residual plots. A two-tailed p value of <0.05 was considered significant.

Results

[0161] Efficacy of S.sup.2+SSCs in Enhancing Recovery from Ventilation Induced ALI

[0162] 40 animals were entered into the experimental protocol, with 10 allocated to each of the VILI groups. Four VILI animals, two allocated to receive vehicle, and two allocated to receive fibroblasts, did not survive the injury protocol. All other animals survived the injury protocol and subsequent treatment allocation. 8 animals each were entered into the vehicle control and fibroblast groups, while 10 animals each received hSSCs and S2.sup.+SSCs.

[0163] Baseline Characteristics: There were no differences among the VILI groups at baseline in terms of pre-injury variables, the duration of injurious ventilation or the extent of the lung injury produced (Table 9).

TABLE-US-00009 TABLE 9 Baseline data regarding animals subjected to high stretch Ventilation. High Stretch Ventilation Variable Vehicle Fibroblasts hSSCs S2.sup.+SSCs Number of animals 8 8 10 10 Animal Weight (g) 400 ± 26  392 ± 51  410 ± 19  417 ± 18  Ventilation Time (mins) 76 ± 27 76 ± 16 77 ± 19 78 ± 14 Lung compliance Pre-Injury (ml/mmHg 0.64 ± 0.09 0.66 ± 0.12 0.67 ± 0.13 0.66 ± 0.11 Lung compliance post-VILI 0.31 ± 0.02 0.32 ± 0.02 0.31 ± 0.03 0.32 ± 0.03 Note: Data are expressed as mean ± SD.

[0164] S2.sup.+SSCs restored lung function and structure following VILI: S2.sup.+SSC therapy enhanced restoration of arterial oxygenation, as evidenced by a reduced alveolar-arterial oxygen gradient compared to vehicle (p<0.05). Further functional recovery in lung physiology in response to S2.sup.+SSC therapy was demonstrated by significant improvements (p<0.01) in respiratory system static compliance in comparison to vehicle.

[0165] S2.sup.+SSCs improved lung microvascular permeability, as evidenced by a decrease in lung wet:dry weight ratios and a decrease in alveolar fluid protein concentrations (Table 10). hSSCs enhanced recovery of lung structure. S2.sup.+SSCs decreased alveolar thickening, as evidenced by reduced alveolar tissue volume fraction, and increased recovery of airspace volume, as evidenced by increased alveolar air-space volume fraction (Table 10).

TABLE-US-00010 TABLE 10 Data regarding extent of resolution 24 hours following high stretch Ventilation. High Stretch Ventilation Variable Vehicle Fibroblasts hSSCs S2.sup.+SSCs Arterial O.sub.2 tension (FiO.sub.2 = 0.3; KPa) 13.4 ± 2.8  12.7 ± 2.8  16.9 ± 2.9*  17.0 ± 1.7*  Arterial O.sub.2 tension (FiO.sub.2 = 1.0; KPa) 32.1 ± 13.1 32.8 ± 16.0 65.3 ± 9.4*  56.2 ± 14.4* Lung Static Compliance (ml/mmHg) 0.37 ± 0.04 0.34 ± 0.08 0.55 ± 0.14* 0.53 ± 0.08* Lung Wet Dry weight ratios 5.9 ± 0.8 5.4 ± 0.9 4.6 ± 0.2* 4.3 ± 0.7* Note: Data are expressed as mean ± SD. Final data is data collected upon completion of the experimental protocol. *Significantly different vehicle and fibroblast groups.

[0166] S2.sup.+SSCs modulated inflammation following VILI: S2.sup.+SSCs decreased total inflammatory cell counts in BAL (bronchoalveolar lavage) fluid and substantially attenuated (p<0.001) lung neutrophil accumulation. Both S2.sup.+SSCs and undifferentiated hSSCs were equally effective in modulating the inflammatory response following VILI (Table 11).

TABLE-US-00011 TABLE 11 Data regarding the inflammatory response 24 hours following high stretch Ventilation. High Stretch Ventilation Variable Vehicle Fibroblasts hSSCs S2.sup.+SSCs BAL 2.91 ± 1.0  3.42 ± 0.86 1.30 ± 0.32* 1.50 ± 0.51* Cell Counts (×10   /ml) % BAL Neutrophils (%) 44.7 ± 12.2 56.7 ± 3.4  15.8 ± 8.5*  16.0 ± 8.5*  BAL Neutrophil Counts (×10   /ml) 1.31 ± 0.60 1.92 ± 0.44 0.20 ± 0.10* 0.27 ± 0.22* BAL Lymphocyte Counts (×10   /ml) 1.57 ± 1.02 0.94 ± 0.44 0.57 ± 0.14† 1.03 ± 0.67  Note: Data are expressed as mean ± SD. Final data is data collected upon completion of the experimental protocol. *Significantly different vehicle and fibroblast groups. †Significantly different from vehicle Group indicates data missing or illegible when filed

[0167] Effect on ‘non-injury’ parameters: There was no effect of S2.sup.+SSCs or undifferentiated hSSCs on arterial pH, PCO.sub.2, bicarbonate, base excess, lactate or mean arterial pressure (data not shown).

Effect of S2.SUP.+.SSCs in Animals Following Low Stretch Ventilation

[0168] 16 animals were entered into the experimental protocol, with 4 allocated to each of the groups. All animals survived the injury protocol and subsequent treatment allocation.

[0169] Baseline Characteristics: There were no differences among the protective ventilation groups at baseline in terms of pre-injury variables, the duration of injurious ventilation or the extent of the lung injury produced (data not shown).

[0170] S2.sup.+SSCs did not affect lung function or structure: There was no effect of S2.sup.+SSC therapy on lung structure or function following protective ventilation (Table 12).

TABLE-US-00012 TABLE 12 Data regarding extent of resolution 24 hours following low stretch Ventilation. Low Stretch Ventilation Variable Vehicle Fibroblasts hSSCs S2.sup.+SSCs Arterial O.sub.2 tension (FiO.sub.2 = 0.3; KPa) 17.6 ± 1.2  17.8 ± 0.8  17.8 ± 0.6  18.5 ± 0.7  Arterial O.sub.2 tension (FiO.sub.2 = 1.0; KPa) 65.8 ± 1.7  69.2 ± 1.7  68.8 ± 3.3  64.3 ± 6.3  Lung Static Compliance (ml/mmHg) 0.53 ± 0.03 0.59 ± 0.06 0.64 ± 0.02 0.61 ± 0.04 Lung Wet:Dry weight ratios 4.3 ± 0.4 4.3 ± 0.5 4.2 ± 0.2 4.3 ± 0.6 Note: Data are expressed as mean ± SD. Final data is data collected upon completion of the experimental protocol.

[0171] S2.sup.+SSCs did not cause inflammation: There was no effect of S2+SSCs therapy on the inflammatory response in the lung structure following protective ventilation (Table 13).

TABLE-US-00013 TABLE 13 Data regarding the inflammatory response 24 hours following low stretch Ventilation. Low Stretch Ventilation Variable Vehicle Fibroblasts hSSCs S2.sup.+SSCs BAL Cell Counts (×10   /ml) 1.24 ± 0.24 1.08 ± 0.13 1.01 ± 0.10 1.14 ± 0.32 % BAL Neutrophils (%) 11.3 ± 2.8  9.8 ± 2.1 20.8 ± 4.9  10.3 ± 2.0  BAL Neutrophil Counts (×10   /ml) 0.14 ± 0.06 0.10 ± 0.02 0.21 ± 0.04 0.11 ± 0.03 BAL Lymphocyte Counts (×10   /ml) 0.64 ± 0.16 0.65 ± 0.38 0.65 ± 0.52 0.59 ± 0.31 Note: Data are expressed as mean ± SD. Final data is data collected upon completion of the experimental protocol. indicates data missing or illegible when filed

[0172] Effect on ‘non-injury’ parameters: There was no effect of S2.sup.+SSCs or undifferentiated hSSCs on arterial pH, PCO.sub.2, bicarbonate, base excess, lactate or mean arterial pressure (Table 14).

TABLE-US-00014 TABLE 14 Data regarding ‘non-injury’ parameters 24 hours following low stretch Ventilation Low Stretch Ventilation Variable Vehicle Fibroblasts hSSCs S2.sup.+SSCs Arterial pH 7.40 ± 0.04 7.39 ± 0.03 7.38 ± 0.03 7.40 ± 0.04 Arterial PCO.sub.2 (KPa) 5.4 ± 0.8 5.5 ± 0.2 5.0 ± 0.2 4.4 ± 0.3 Arterial Bicarbonate (mMol/L) 20.5 ± 2.0  22.0 ± 1.5  20.9 ± 1.0  21.7 ± 2.1  Base Excess 3.4 ± 1.5 3.3 ± 1.7 3.4 ± 2.0 2.8 ± 1.8 Arterial Lactate (mMol/L) 3.1 ± 1.4 2.2 ± 0.6 2.1 ± 0.8 2.0 ± 1.2 Mean Arterial Pressure (mmHg) 113.2 ± 2.7  101.0 ± 10.7  98.0 ± 13.7 99.5 ± 17.1 Note: Data are expressed as mean ± SD. Final data is data collected upon completion of the experimental protocol.

CONCLUSIONS

[0173] S2.sup.+SSCs of the invention restored lung function and structure following VILI, as evidenced by a reduced alveolar-arterial oxygen gradient, significant improvements (p<0.01) in respiratory system static compliance, and improved lung microvascular permeability. Also, they enhanced recovery of lung structure following VILI. The cells modulated inflammation following VILI, decreasing total inflammatory cell counts in BAL fluid and substantially attenuating (p<0.001) lung neutrophil accumulation. There was no effect of S2.sup.+SSC therapy on lung structure or function, or on the inflammatory response, following protective ventilation. These findings suggest that the cells of the invention are well tolerated in this model.

[0174] The invention thus provides methods of obtaining defined stromal stem cell populations and uses thereof.