ORIGINAL ARTICLES Isolation of functionally distinct mesenchymal stem cell subsets using antibodies against CD56, CD271, and mesenchymal stem cell antigen-1

Venkata Lokesh Battula,1* Sabrina Treml,1 Petra M. Bareiss,2 Friederike Gieseke,3 Helene Roelofs,4 Peter de Zwart,5 Ingo Müller,3 Bernhard Schewe,5 Thomas Skutella,2 Willem E. Fibbe,4 Lothar Kanz,3 and Hans-Jörg Bühring1

1University Clinic of Tübingen, Department of Internal Medicine II, Division of Hematology, Oncology, and Immunology, Tübingen; 2University of Tübingen, Institute of Anatomy, Department of Experimental Embryology, Division of Tissue Engineering, Tübingen; 3University Clinic of Tübingen, Children's Hospital, Department of General Pediatrics, Division of Hematology and Oncology, Tübingen; 4Leiden University Medical Center, Department of Immunohematology and Blood Transfusion, Center for Stem Cell Therapy, Leiden, The Netherlands, and 5Hospital for Workers Compensation Tübingen, Department of Orthopedic Surgery, Tübingen, Germany

ABSTRACT *present address: MD Anderson Cancer Center, Background Houston, TX, USA. Conventionally, mesenchymal stem cells are functionally isolated from primary tissue VLB, ST and H-JB contributed based on their capacity to adhere to a plastic surface. This isolation procedure is hampered equally to this work. by the unpredictable influence of co-cultured hematopoietic and/or other unrelated cells and/or by the elimination of a late adhering mesenchymal stem cells subset during Acknowledgments: the authors removal of undesired cells. To circumvent these limitations, several antibodies have been would like to thank Flavianna Cerabona for excellent developed to facilitate the prospective isolation of mesenchymal stem cells. Recently, we technical assistance. described a panel of monoclonal antibodies with superior selectivity for mesenchymal stem cells, including the monoclonal antibodies W8B2 against human mesenchymal stem Funding: this work was cell antigen-1 (MSCA-1) and 39D5 against a CD56 epitope, which is not expressed on nat- supported by the Deutsche ural killer cells. Forschungsgemeinschaft (DFG), Sonderforschungsbereich Design and Methods SFB-685 (Immunotherapy: Molecular Basis and Clinical Bone marrow derived mesenchymalFoundation stem cells from healthy donors were analyzed and Applications) project Z2: Core isolated by flow cytometry using a large panel of antibodies against surface antigens Facility Cell Sorting, by the including CD271, MSCA-1, and CD56. The growth of mesenchymal stem cells was mon- DFG project BU 516/2-1: itored by colony formation unit fibroblast (CFU-F) assays. The differentiation of mes- Identifizierung und funktionelle Untersuchung von MSC-spezifis- enchymal stem cells into defined lineages was induced by culture in appropriate media chen Molekülen, and by the and verified by immunostaining.Storti Federal Ministry for Education and Research (BMBF), Results BioProfile Stuttgart/Neckar-Alb; Multicolor cell sorting and CFU-F assays showed that mesenchymal stem cells were ~90- project 0313668B: fold enriched in the MSCA-1+CD56− fraction and ~180-fold in the MSCA-1+CD56+ frac- Entwicklung eines bioartifiziellen Leberreaktors mit allogenen tion. Phenotype analysis revealed that the expression of CD10, CD26, CD106, and CD146 humanen Hepatozyten. was restricted to the MSCA-1+CD56− mesenchymal stem cells subset and CD166 to MSCA-1+CD56± mesenchymal stem cells. Further differentiation of these subsets showed Manuscript received August 1, that chondrocytes and pancreatic-like islets were predominantly derived from MSCA- 2008. Revised version arrived ©Ferrata1+CD56± cells whereas adipocytes emerged exclusively from MSCA-1+CD56− cells. The September 10, 2008. + + Manuscript accepted culture of single sorted MSCA-1 CD56 cells resulted in the appearance of phenotypically September 24, 2008. heterogeneous clones with distinct proliferation and differentiation capacities. Correspondence: Conclusions Hans-Jörg Bühring, Ph.D., Novel mesenchymal stem cells subsets with distinct phenotypic and functional properties University of Tübingen, were identified. Our data suggest that the MSCA-1+CD56+ subset is an attractive starting Department of Internal Medicine II, Medical population for autologous chondrocyte transplantation. Otfried-Müller-Str. 10, 72076 Tübingen, Germany. Key words: mesenchymal stem cells, CD56, MSCA-1. E-mail: hans-joerg.buehring@uni-tuebin- Citation: Battula VL, Treml S, Bareiss PM, Gieseke F, Roelofs H, de Zwart P, Müller I, Schewe B, gen.de Skutella T, Fibbe WE, Kanz L, and Bühring H-J. Isolation of functionally distinct mesenchymal stem cells subsets using antibodies against CD56, CD271, and mesenchymal stem cell antigen-1. Haematologica 2009; 94:173-184. doi: 10.3324/haematol.13740

©2009 Ferrata Storti Foundation. This is an open-access paper.

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Introduction Design and Methods

Mesenchymal stem/stromal cells (MSC) are self- Isolation of bone marrow and peripheral blood renewing cells with the ability to differentiate into mononuclear cells osteocytes, chondrocytes and adipocytes, but also neu- Bone marrow was harvested at the Hospital for ron-like cells, hepatocytes and pancreatic-like cells.1-4 Workers Compensation from the femoral shafts of These multipotent cells are found in various adult and patients undergoing total hip replacement. Peripheral fetal tissues including bone marrow, umbilical cord blood from healthy volunteers was obtained from the blood, liver, dental pulp, and term placenta.5-13 In cul- Transfusion Department, Tübingen. Cells were collected ture, expanded MSC express a panel of key markers in 5000 U heparin (Sigma-Aldrich, Taufkirchen, Germany) including CD105 (, SH2), CD73 (ecto-5’ after informed consent and approval of the ethics commit- nucleotidase, SH3, SH4), CD166 (ALCAM), CD29 (β1- tee of the University of Tübingen. Bone marrow mononu- ), CD44 (H-CAM), and CD90 (Thy-1).1-3,7,14 In clear cells and peripheral blood mononuclear cells were contrast to hematopoietic stem cells they lack CD45, isolated by Ficoll Histopac density gradient fractionation CD34, and CD133 expression.1,2,15 Because of their and remaining erythrocytes lysed in ammonium chloride multi-lineage differentiation potential, MSC represent solution.5 an attractive cell type for replacement therapy of dam- aged tissues.16-18 Culture of primary cells MSC can be identified by their ability to form colony Ficoll-separated and FACS-enriched bone marrow forming units-fibroblast (CFU-F) in vitro.2,7 However, mononuclear cells were cultured as previously described.5 these cells are heterogeneous with respect to their pro- In brief, 2×107 unfractionated or 1×104 sorted MSCA- liferation and differentiation capacity. At least two mor- 1+CD56+ and MSCA-1+CD56- bone marrow cells were phologically distinct MSC populations have been iden- cultured in gelatine-coated T-75 or T-25 culture flasks in tified that differ not only in size but also in their cell the presence of KnockoutTM medium (Invitrogen, division rate and differentiation capacity.4 In addition, Karlsruhe, Germany) and 5 ng/mL recombinant human analysis of single cell-derived MSC colonies from adult basic fibroblast growth factor (rh-bFGF; CellSystems, bone marrow revealed differential capacity of colonies Remagen, Germany).32 After 3 days of culture, non-adher- to undergo osteogenic, adipogenic, and chondrogenic ent cells were removed and fresh medium was added. differentiation.4,19 AdherentFoundation cells were cultured until they reached 90% con- In most cases, unfractionated bone marrow-derived fluence. cells are used as the starting population for the culture of MSC. This isolation method relies on the adherence Colony forming unit-fibroblast assay of fibroblast-like cells to a plastic surface and the CFU-F assays were performed by plating either 1×105 removal of non-adherent hematopoietic cells.1-3,7,15,17StortiThe unselected or 500-5,000 FACS-selected bone marrow resulting cells are poorly defined and give rise not only mononuclear cells in gelatine-coated T-25 flasks contain- to heterogeneous MSC populations but also to ing KnockoutTM medium and 5 ng/mL rh-bFGF. After 12 osteoblasts and/or osteoprogenitor cells, fat cells, retic- days of culture, adherent cells were washed twice with ular cells, macrophages, and endothelial cells.20,21 To phosphate-buffered saline, fixed with methanol (Sigma- define the starting population more precisely, surface Aldrich) for 5 min at room temperature, air-dried, and markers such as SH2 (CD105), SH3/SH4 (CD73), SSEA- stained with Giemsa solution (Merck, Darmstadt, 4, STRO-1 and the low affinity nerve growth factor Germany). CFU-F colonies were macroscopically enu- receptor (CD271), which enrich for MSC, have been merated. Colony sizes ranged between 1 and 8 mm in employed.21-25 Very recently, platelet derived growth diameter. factor receptor-β (PDGF-RB;©Ferrata CD140b) was identified as a selective marker for the isolation of clonogenic MSC.26 Differentiation of mesenchymal stem cells Other reports demonstrated a 9.5-fold enrichment of Osteoblast and adipocyte differentiation MSC in bone marrow cells with prominent aldehyde MSC derived from sorted MSCA-1+CD56+ or unfrac- dehydrogenase activity.27 tionated bone marrow cells were cultured in NH Recently, we have shown that CD56, a marker for OsteoDiff or NH AdipoDiff medium (Miltenyi Biotec, natural killer, neural, and muscle cells,28-30 is additionally Bergisch Gladbach, Germany), respectively.5 In brief, expressed on a small subset of bone marrow CD271bright 2×104 (osteogenesis) or 4×104 (adipogenesis) MSC were cells.26 We also demonstrated that W8B2 antigen31 (here cultured in 24-well Falcon plates (Becton Dickinson, designated mesenchymal stem cell antigen-1; MSCA-1) Heidelberg, Germany). After 12 days of culture in NH is selectively expressed on CD271bright bone marrow cells OsteoDiff medium, cells were fixed with methanol (- and hypothesized that W8B2 antibody reacts with 20°C, 5 min). Alkaline phosphatase activity in osteoblasts CFU-F, as these clonogenic cells are known to reside in was determined using FASTTM BCIP/NBT substrate the CD271bright population.26 In this work we investigat- (Sigma-Aldrich). Calcium deposition in fixed cells (4% ed the composite phenotype and expression pro- paraformaldehyde, 15 min) was analyzed after staining file, as well as the clonogenic, differentiation, and with 2% alizarin red S (Merck) for 10 min at room tem- immunomodulation potential of MSCA-1+ and CD56+ perature. The formation of adipocytes was evaluated after MSC populations in bone marrow. 25 days of culture in NH AdipoDiff medium and staining

| 174 | haematologica | 2009; 94(2) Novel MSC markers of methanol-fixed cells with oil red O dye (Sigma- Pancreatic differentiation Aldrich) for 45 min at room temperature. Pictures were MSC (5×105) were plated into 6-well ultra low taken using an Axiovert 40C microscope (Carl Zeiss adherent dishes (Costar; CellSystems) and cultured for GmbH, Göttingen, Germany). 4 days in minimal essential medium containing 1 mM mono-thioglycerol, 15% ES-Cult fetal bovine serum, Chondrogenic differentiation and 4.5 g/L DMEM high glucose (CellSystems). The MSC (4×105) were cultured as a pellet for 4 hours in resulting cell clusters were then cultured for 6 days in 15 mL Falcon tubes (Becton Dickinson) at 37°C in 20 µL 6-well adherence plates (Falcon) in ITS supplemented of Incomplete Chondrogenic Induction Medium (PAA, serum-free medium (CellSystems). After transfer into Pasching, Austria) containing 1% ITS-supplements poly-L-ornithine coated 24-well plates, cells were cul- (Sigma-Aldrich), 175 µM L-ascorbic acid (Sigma- tured for 6 days in pancreatic proliferation medium Aldrich), 350 µM L-proline (Sigma-Aldrich), and 100 (CellSystems) containing N2-A and B27 supplements nM dexamethasone (Sigma-Aldrich). After incubation, and 25 ng/mL rh-bFGF, and then for 6 more days in 10 400 µL of Complete Chondrogenic Induction Medium mM nicotinamide containing rh-bFGF-free pancreatic supplemented with 10 ng/mL of transforming growth differentiation medium (CellSystems). After washing, factor β (Sigma-Aldrich) were added. The resulting cell cells were fixed with 4% phosphoformic acid, perme- pellets were cultured for 3 weeks, fixed with 4% phos- abilized with 70% ethyl alcohol, and incubated with phoformic acid, embedded in paraffin, and cut into 5 blocking buffer containing 0.25% Triton X-100 and µm thick sections. The dried and deparaffinized sec- 2% fetal bovine serum, labeled with polyclonal rabbit tions were incubated with Alcian blue solution (Merck) anti-human glucagon antibody (1:75 dilution; Dako for 45 min at room temperature, washed in 3% acetic Cytomations, Glostrup, Denmark) or polyclonal rabbit acid, embedded and photographed on a Zeiss Axiovert anti-human insulin antibody (1:200 dilution; Santa 200 microscope. Cruz Biotechnology) overnight and stained with sec- ondary goat anti-rabbit IgG-Cy3 (Millipore, Schwal- Myogenic differentiation bach, Germany), and 0.4 µg/mL DAPI. MSC (5×105) were cultured for 7 days on ultra low adherent dishes in Dulbecco’s modified Eagle’s medi- Generation of mesenchymal stem cell-reactive mono- um (DMEM) high glucose (Invitrogen), supplemented clonal antibodies, W8B2 and 39D5 with 100 µM β-mercaptoethanol. The resulting embry- The W8B2 monoclonal antibody (IgG1; specificity for oid body-like clusters were incubated in gelatine-coat- hMSCA-1)Foundation was raised by immunization of a 6-8 week- ed 24-well dishes for 21-28 days, the resulting cells old female Balb/c mouse (Charles River WIGA, fixed with 4% phosphoformic acid for 45 mins at room Sulzfeld, Germany) with the retinoblastoma cell line temperature and permeabilized with 0.1% Triton X- WERI-RB-1, as previously described.31 The 39D5 mono- 100/phosphate-buffered saline for 20 min. Cells were clonal antibody (IgG1, CD56) was raised by immuniza- labeled over night at 4°C with rabbit anti-humanStortition with the hematopoietic cell line KG-1a.33 smooth muscle actin antibody (Spring Bioscience, Freemont, CA, USA) and mouse anti-human sarcomer- Immunofluorescence analysis and cell sorting ic actinin (anti α-actinin) antibody (Sigma-Aldrich). Antibodies After washing, cells were stained with Cy3-conjugated The following proprietary antibodies were used: goat anti-rabbit IgG (Jackson Immuno Research), or 97C5 (CD10),34 46A11 (CD13),35 39D5 (CD56),33 1G2C2 Alexa Fluor488-conjugated goat anti-mouse IgG (CD105),31 104D2 (CD117),34 W6B3C1 (CD133),31 28D4 (Invitrogen), and 0.4 µg/mL 4’6 diamidino-2 phenylin- (CD140b),26 67D2 (CD164),37,38 CUB1 (CD318; dole (DAPI). CDCP1),39 24D2 (CD340; HER-2),26 W3C4E11 (CD349; frizzled-9),5 HEK-3D6 (unknown),26 W1C3 (unknown),26 Neuronal differentiation ©Ferrata W5C4 (unknown),26 W5C5 (unknown),26 W3D5 MSC (3.5×104) were cultured for 6 days in 800 µL (unknown),26 and W8B2B10 (MSCA-1).31 CD34-PE NeuroCult® NS-A proliferation medium (CellSystems) (clone 8G12), CD45-PE (clone HI30), CD56-FITC (clone and then for 7 days in NeuroCult® NS-A differentiation NCAM16.2), CD56-PE (clone NCAM16.2), CD90-APC medium (CellSystems). Cells were fixed with 4% (5E10), CD63-PE (clone H5C6), CD73-PE (clone AD2), phosphoformic acid and permeabilized with 0.3% and HLA-DR-PE (clone TÜ36) were purchased from Triton-X-100/phosphate-buffered saline (Sigma- Becton Dickinson (Heidelberg, Germany). The SSEA-4 Aldrich) prior to incubation overnight with rabbit anti- reactive antibody MC-813-70 was purchased from human glial fibrillary acidic antibody or mouse Chemicon (Hampshire, UK). CD271-APC (clone anti-human neuronal class III-β-tubulin antibody (both ME20.4-1.H4) was obtained from Miltenyi Biotec. reagents from CellSystems). CD105-PE (clone SN6) was purchased from eBioscience After washing with 0.1% BSA/TBS/Tween-20 Inc. (San Diego, CA, USA). CD166-PE was a kind gift (Sigma-Aldrich), cells were stained with Cy3-conjugat- from Dr. Gene Lay (BioLegend, San Diego, CA, USA). ed porcine anti-rabbit secondary antibody (Jackson Immuno Research, Cambridge, UK), for 30 min at Immunofluorescence staining room temperature or Alexa Fluor® 488-conjugated goat After blocking of non-specific binding with 10 anti-mouse IgG secondary antibody (Invitrogen), and mg/mL polyglobin (10 min, 4°C), cells were incubated 0.4 µg/mL DAPI. for 15 min with either 20 µL of proprietary antibodies

haematologica | 2009; 94(2) | 175 | V.L. Battula et al.

or 10 µL of fluorochrome-conjugated antibodies.5 Cells Gene chip analysis of sorted cells stained with conjugates were washed twice, suspended Ten thousand MSCA-1+CD56– and MSCA-1+CD56+ in 200 µL FACS buffer and used for flow cytometry. cells were used for commercial gene chip analysis Cells labeled with proprietary antibodies were stained (Miltenyi Biotec) to perform human whole genome with 20 µL of a F(ab)2 fragment of a R-phycoerythrin oligo microarray (Agilent Technologies, Böblingen, (PE) conjugated goat anti-mouse antibody (Dako Germany). Amplified cDNA were quantified using a Cytomations, Glostrup, Denmark) for 15 min, washed ND-1000 spectrophotometer (NanoDrop Technologies twice and analyzed by flow cytometry. For multi-color Inc, Wilmington, DE, USA). Two hundred and fifty staining, cells were incubated for 15 min with 10 µL of nanograms of the library polymerase chain reactions anti-CD56-FITC and anti-CD271-APC and/or the indi- were used as the template for Cy3 and Cy5 labeling. cated PE-conjugate. Mouse IgG1 antibodies conjugated The samples were hybridized for 17 h at 65°C on with FITC, PE, or APC (Becton Dickinson) were used as Agilent’s microarray according to the manufacturer’s isotype controls. After washing, cells were used for instructions. Gene chip scanning and data analysis were flow cytometry. For combined indirect and direct stain- carried out using Luminator software (Miltenyi Biotec). ing, cells were first labeled with the non-conjugated antibody and then stained with 20 µL of 1:25 diluted Statistics goat anti-mouse secondary antibody for 15 min. Free Statistical analysis was performed using Student’s t- binding sites of the secondary antibody were blocked test and a p-value less than 0.05 was considered statisti- by incubating cells for 25 min with 20 µL mouse IgG cally significant. polyclonal antibody (0.05 µg/mL; Southern Biotech, Birmingham, AL, USA) prior to counter-staining with CD271-APC and/or CD56-FITC. For controls, second- Results ary step antibody conjugates were used in addition to IgG1-FITC and IgG1-PE. After washing, cells were ana- The 39D5 monoclonal antibody detects a rare bone lyzed by flow cytometry. marrow CD271+ subset Recently, we showed that the CD56-reactive 39D5 Flow cytometric analysis and cell sorting monoclonal antibody recognizes a rare subset of Cells were sorted on a FACSAria cell sorter (Becton CD271bright cells.26 Notably, 39D5 recognizes an epitope Dickinson), or analyzed on a FACSCantoII flow of CD56 not expressed on the surface of peripheral cytometer (Becton Dickinson). Files were analyzed blood-derivedFoundation natural killer cells.33 Indeed, comparative using the FCS express software (De Novo Software, flow cytometric analysis showed that only the commer- Ontario, Canada). Single cell sorting in 96-well plates cially available CD56-specific antibody NCAM16.2 but was performed using the ACDU device. not the 39D5 monoclonal antibody reacted with 20±10% of peripheral blood cells (Figure 1A). In contrast Magnetic-activated cell-separation Stortito the situation with peripheral blood cells, both mono- For some experiments, bone marrow cells were pre- clonal antibodies reacted with a small subpopulation selected by MACS (Miltenyi Biotec) using CD271-APC (0.5-5.5%) of bone marrow CD271bright cells (Figure 1B). and anti-APC beads. The cells were separated according Simultaneous staining of bone marrow cells with 39D5 to the manufacturer’s recommendations. and NCAM16.2 revealed that both antibodies detected the same CD271bright population (data not shown). Inhibition of T-cell proliferation HLA-mismatched peripheral blood mononuclear cells The CD271brightCD56+ population is enriched for CFU-F were labeled with 0.8 µM carboxyfluorescein diacetate To determine the clonogenic potential of sorted succinimidyl ester (CFSE; Invitrogen) and cultured at CD56+ and CD56– subsets, CFU-F assays were per- 75,000 cells/well for 4 days©Ferrata in 96-well plates (Greiner , formed. Figure 1C shows a 3 (±0.8)-fold enriched Frickenhausen, Germany) coated with either 10,000, cloning efficiency of CD271brightCD56+ cells compared to 20,000 or 30,000 unfractionated or sorted MSC. Cells CD271brightCD56– cells and a 180 (±52) fold enrichment of were cultured in RPMI-1640 (Biochrom) supplemented CFU-F compared to unfractionated bone marrow cells. with either 100 U/mL interleukin-2 (R&D Systems, The enrichment was independent of the analyzed CD56 Minneapolis, MN, USA) and 1 µg/mL OKT3 antibody epitope (data not shown). Interestingly, CD271brightCD56+ (Janssen-Cilag, Neuss, Germany), or with 3 µg/mL phy- cells did not only give rise to higher colony numbers tohemogglutinin (Sigma-Aldrich). After incubation, (38/500 versus 12/500 plated cells) but were also 2 to 4- cells were analyzed by flow cytometry. fold enriched in very large colonies (>100 cells/colony; data not shown). Inhibition of dendritic cell differentiation One million freshly isolated monocytes (CD14 Phenotype of mesenchymal stem cells derived from MicroBeads, Miltenyi Biotec) were cultured with inter- sorted CD271brightCD56+/– bone marrow cells leukin-4 (10 ng/mL) and granulocyte-monocyte colony- CD271brightCD56+ and CD271brightCD56– bone marrow stimulating factor (5 ng/mL) in the presence or absence cells were separated by FACS, cultured in gelatine-coat- of 105 MSC. After 6 days of culture, cells were assayed ed flasks in the presence of serum replacement medium by flow cytometry for CD14 (Becton Dickinson) and (n=3),5 stained with the indicated antibodies and ana- CD1a (Becton Dickinson) expression.40 lyzed by flow cytometry. Figure 1D shows that CD10,

| 176 | haematologica | 2009; 94(2) Novel MSC markers

A

CD56 (NCAM16.2) CD56 (E-39D5)

B CD271

CD56 (NCAM16.2) CD56 (E-39D5) 50 C 40 30

CFU-F 20

10 0 Unsorted CD271+ CD271+ CD56– CD56+ CD271+CD56− CD271+CD56+ Figure 1. Characterization of CD56+ bone marrow cells. (A) CD56 epitope NCAM16.2 but not 39D5 is expressed on peripheral blood natural killer CD10 CD56 CD105 CD117 CD10 CD56 CD105 CD117 cells. (B) CD56 epitopes NCAM16.2 and 39D5 are expressed on a rare CD271+ bone marrow subset. (C) CD271+CD56+ and CD271+CD56– D CD140b CD271 CD318 CW8B2 CD140b CD271 CD318 CW8B2 bone marrow cells are clonogenic. CFU-F derived Foundationfrom 500 FACS-sorted cells were stained and scored as described. Data represent the mean SSEA-4 HEK-3D6 HER-2 FZD9 SSEA-4 HEK-3D6 HER-2 FZD9 CFU-F numbers of three different experiments (*=p<0.01). (D) Expression of selected markers on cultured CD271+CD56– and CD271+CD56+ derived mesenchymal stem cells. W1C3 W5C4 W5C5 W3D5 W1C3Storti W5C4 W5C5 W3D5 CD140b, CD318, HER2 (CD340), frizzled-9 (CD349), class II DM β, spondin 2, and HLA class II DR α were as well as the antibody-defined antigens W1C3, W5C4, expressed at 62 to 23-fold decreased levels in this subset W5C5, and W3D5 (and CD166; data not shown) were (data not shown), indicating a high diversity of gene similarly expressed on MSC derived from both frac- expression profiles in these subsets. tions. CD271, SSEA-4 and CD56 were more densely expressed on CD271brightCD56+ derived MSC, whereas Phenotype of CD271brightCD56+/– bone marrow cells MSCA-1 (W8B2 antigen) expression was more pro- To compare the expression profiles of surface markers nounced on CD271brightCD56– derived MSC. In contrast on CD271brightCD56+ and CD271bright CD56– cells, bone to primary MSC (Figure©Ferrata 2B), cultured MSC de novo marrow cells were triple-stained with anti-CD271, anti- expressed CD166 and CD318 (and CD109; data not CD56 and a panel of test antibodies and gated on the shown) and downregulated CD271 expression. CD271bright population (Figure 2A; gate R1). Figure 2B shows that CD63, CD73, CD140b, CD164, and W3D5 Gene expression analysis of primary CD271brightCD56– antigen were expressed at similar levels on both cell and CD271brightCD56+ bone marrow cells subsets, whereas CD45, CD117, CD133, CD318 were Whole genome microarray analysis of 10,000 sorted negative. In contrast, CD271bright CD56+ cells expressed bone marrow cells was performed to compare the gene CD13, CD105, frizzled-9 (CD349), HLA-DR, and expression profile of CD271brightCD56– and CD271bright MSCA-1 (W8B2 antigen) at reduced levels, whereas CD56+ bone marrow cells. CD271brightCD56+ cells CD166 was found exclusively on these cells. The fact showed an 11 to 43 fold increased expression of secret- that CD166 expression is absent on the majority of pri- ed frizzled-related protein 4, esophageal cancer related mary MSC is surprising because cultured MSC are gene 4 protein, carboxypeptidase E, platelet-derived known to express high levels of CD166. Likewise, the growth factor A, eukaryotic translation termination fac- tumor antigen CDCP1 (CD318) was not expressed on tor 1, and CD163 (data not shown). In contrast, primary CD271bright CD56– and CD271brightCD56+ cells coding for leukocyte immunoglobulin-like receptor sub- (Figure 2B) but highly expressed on cultured MSC family B, zinc finger protein 212, amphiregulin, HLA (Figure 1D).

haematologica | 2009; 94(2) | 177 | V.L. Battula et al.

B A CD13 CD45 CD63 CD73 CD105 R1

CD271 CD117 CD133 CD140b CD164 CD166 SSC

CD138 FZD9 HLA-DR W8B2 W3D5

CD56 Figure 2. Phenotype, clonogenic potential, 80 and morphology of CD271brightMSCA-1+CD56- R2 70 CFU-F 1bright + + 60 and CD27 MSCA-1 CD56 bone marrow 50 cells. Triple-stained bone marrow cells were C D 40 gated on the CD271+ subset and analyzed W8B2 30 for coexpression of CD56 and selected mark- R3 20 ers. (A) Display of SSC versus CD271-APC of 10 0 bone marrow cells. (B) Display of CD56 ver- CD56 Unsorted CD271+ CD271+ sus the indicated markers on CD271bright W8B2++ W8B2+ gated cells. (C) Display of CD56 versus CD56– CD56+ MSCA-1 (W8B2) on CD271bright gated cells. Sort windows are indicated as R2 and R3. E (D) CFU-F numbers derived from 1.000 FACS- sorted bone marrow MSCA-1+CD56- and MSCA-1+ CD56+ cells or 100.000 unfraction- ated bone marrow cells. The resulting CFU-F were stained and scored 12 days after cul- ture and normalized to 1,000 plated cells (p<0.01). (E) Morphology of bone marrow MSCA-1+CD56- and MSCA-1+CD56+ cells. Subsets were sorted, cytocentrifuged, Foundationstained with May-Grünwald-Giemsa solution, and scored on a Zeiss Axiovert 200 micro- scope (Carl Zeiss Inc, Göttingen, Germany). Note the presence of basophilic-like granules in cells of the triple-positive population. CD271+W8B2++CD56– CD271+W8B2+CD56+ Storti Clonogenic capacity of MSCA-1+CD56+/– bone marrow until they had undergone approximately 9-10 cell divi- cells sions. Defined numbers of the resulting MSC were then We recently demonstrated that MSCA-1 (W8B2 anti- induced to differentiate into cells of the osteogenic, adi- gen) expression is restricted to CD271bright cells and that pogenic, chondrogenic, myogenic, neuronal, and pancre- clonogenic MSC reside in the CD271bright but not in the atic lineages. CD271dim population.26 Here we show that MSCA-1 is expressed at high levels on CD271brightCD56– cells and at Osteoblastic differentiation lower levels on CD271brightCD56+ cell subsets (Figure 2A). Culture of MSC (n=5) derived from sorted cell subsets The percentage of CD56+ ©Ferratacells in the MSCA-1+ popula- in appropriate medium resulted in the appearance of tion ranged from 1-20% (21 tested samples; average fre- 95±5% (CD56+) and 70±5% (CD56–) of alkaline phos- quency of CD56+ cells: 8.5%). To analyze the clonogenic phatase-positive cells (Figure 3). In contrast, MSC capacity of these subsets, cells were fractionated accord- derived from unfractionated bone marrow cells gave rise ing to sort windows R2 and R3 (Figure 2C). Defined cell to only 35±5% alkaline phosphatase-positive cells. numbers were plated into culture flasks and the resulting Similarly, alizarine red S staining was observed in all CFU-F enumerated after 12 days of culture. Figure 2D osteoblast fractions. However, the number of calcium shows that MSCA-1+CD56+ cells gave rise to 2±0.4 fold nodules was 2-fold lower in osteoblasts derived from higher CFU-F numbers than MSCA-1+CD56– cells. unfractionated cells. Giemsa staining revealed that MSCA-1+CD56– cells con- tain a large, bright cytoplasm with vacuoles, whereas Adipocyte differentiation MSCA-1+CD56+ cells contain a smaller cytoplasm with Culture of unfractionated and MSCA-1+CD56– derived basophilic granules (Figure 2E). MSC (n=5) in adipocyte differentiation medium resulted in the appearance of oil red O incorporating adipocytes. Differentiation capacity of mesenchymal In contrast, MSCA-1+CD56+ derived MSC were unable stem cells derived from MSCA-1+CD56+/– cells to generate adipocytes (Figure 3). MSCA-1+CD56– MSC For differentiation assays, unfractionated or sorted showed a 5±0.5 fold increase of oil red O-positive MSCA-1+CD56–, MSCA-1+CD56+ cells were expanded adipocytes compared to unfractionated cells. Thus, the

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Unfractionated CD56– CD56+ Inihibition of T-cell proliferation A 100 100 ALP IL-2 + OKT3 80 PHA unfractionated 80 unfractionated 60 MSCA-1+CD56–60 MSCA-1+CD56– MSCA-1+CD56+ MSCA-1+CD56+

diff. 40 40 20 20 proliferating Osteoblast

% 0 0 Red S PBMC PBMC+ PBMC+ PBMC+ PBMC PBMC+ PBMC+ PBMC+ Alizarine 1×104 2×104 3×104 1×104 2×104 3×104 MSC MSC MSC MSC MSC MSC B Inihibition of DC proliferation diff.

+ + + –

Adipocyte unfractionated MSCA-1 /CD56 MSCA-1 /CD56 4 4 4 4 Oil Red O 10 10 10 10 3 3 3 3 10 10 10 10 2 2 2 2 10 10 10 10 1 1 CD14 1 1 CD14PE CD14PE CD14PE CD14PE 10 10 10 10 0 0 0 0 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10

10 10 10 10 10 10 10 10 10 10 1010 10 10 10 10 10 10 10 10 10 CD1aFITC CD1aFITC CD1aFITC CD1aFITC diff. CD1a Chondrocyte Alcian Blue Figure 4. Inhibitory effect of MSC subsets on T-cell proliferation and dendritic cell differentiation. (A) Inhibition of T-cell prolifera- tion. MSC derived from MSCA-1+CD56–, MSCA-1+CD56+ and unfrac- tionated bone marrow cells inhibit the interleukin-2 (IL-2) (100 U/mL) + OKT3 (1 µg/mL) or phytohemagglutinin (PHA) (3 µg/mL) induced proliferation of PBMC to a similar extent. (B) Inhibition of diff. +

-actinin dendritic cell differentiation. MSC derived from MSCA-1 CD56, Myogenic + + α MSCA-1 CD56 , and unfractionated bone marrow cells inhibit the interkeukin-4 (IL-4) (10 ng/mL) + granulocyte-monocyte colony- stimulating factor (5 ng/mL) induced differentiation of CD14+ monocytes into CD1a+ dendritic cells. In the absence of MSC, monocytes differentiated into CD1a+ dendritic cells. GFAP diff. Neuronal ed almostFoundation exclusively in the MSCA-1+CD56+ subset, whereas MSCA-1+CD56– pellets contained mainly -tubulin-III Neuronal

β apoptotic cells. MSC from unfractionated cells gave rise to heterogeneous pellet sizes but consistently with fewer viable cells. These data suggest that effective chondrogenesis is restricted to the MSCA-1+CD56+ Glucagon Storti diff. MSC subset. Pancreatic Myogenic differentiation Insulin Culture of MSCA-1+CD56± derived MSC (n=2) in medium designed for differentiation into striated mus- Figure 3. Differentiation potential of mesenchymal stem cells cle cells resulted in the appearance of striated muscle- derived from sorted MSCA-1+CD56– and MSCA-1+CD56+ bone mar- specific β-actinin staining in cells of all fractions (Figure row cells. Cells triple-stained with CD271, MSCA-1 (W8B2), and CD56 were sorted and cultured as described. Expanded mes- 3). In contrast, the smooth muscle-specific marker enchymal stem cells were induced into osteogenic, adipogenic, smooth muscle actin was negative in all fractions (data chondrogenic, myogenic, neurogenic,©Ferrata and pancreatic differentia- not shown). Undifferentiated MSC showed very weak β- tion and stained as described. The resulting cells were pho- tographed with a Zeiss Axiovert 200 microscope. Note the exclu- actinin staining (data not shown). sive presence of adipocytes in the MSCA-1+CD56 subset and the appearance of chondrocytes and pancreatic-like islets in the Neuronal differentiation MSCA-1+CD56+ fraction. MSC (n=3) cultured in neuronal differentiation medium were stained for glial fibrillary acidic protein and neuronal adipocyte differentiation potential is restricted to the β-tubulin-III. Figure 3 shows a prominent staining of cells MSCA-1+CD56– subset. derived from unfractionated as well as from MSCA- 1+CD56+ and MSCA-1+CD56– derived MSC. No staining Chondrogenic differentiation was observed in undifferentiated MSC or in differentiat- To analyze the chondrocyte differentiation potential, ed cells labeled with isotype-matched control antibodies MSC (n=3) derived from fractionated and unfractionat- (data not shown). ed cells were cultured in appropriate medium and the resulting cell pellets stained with Alcian blue. Although Pancreatic differentiation chondrogenic differentiation was detected in both frac- Culture of MSC (n=2) in pancreatic differentiation tions, pellet slices from MSCA-1+CD56+ cells were medium resulted in glucagon and insulin staining of pan- 5±1.6 times larger than those from MSCA-1+CD56– cells creatic-like islets in cells of all fractions (Figure 3). (Figure 3). In addition, viable chondrocytes were detect- However, islets derived from MSCA-1+CD56+ MSC were

haematologica | 2009; 94(2) | 179 | V.L. Battula et al.

larger and the staining intensity of these markers was phytohemogglutinin. Figure 4A shows that MSC from all much more pronounced compared to MSCA-1+CD56– fractions inhibited the proliferation of peripheral blood derived or unfractionated MSC. No staining was observed mononuclear cells without significant differences in a in undifferentiated MSC or in differentiated cells labeled dose-dependent manner and to a similar extent. We also with isotype-matched control antibodies (data not shown). tested the effect of MSC derived from unfractionated and MSCA-1+CD56± populations on the interleukin-4 + gran- Inhibition of T-cell proliferation and dendritic cell ulocyte-monocyte colony-stimulating factor induced dif- differentiation ferentiation of monocytes into dendritic cells (n=3). Figure MSC derived from MSCA-1+CD56± cells were analyzed 4B shows that all MSC subsets were able to inhibit differ- for their capacity to inhibit T-cell proliferation and den- entiation from CD14+ monocytes to CD1a+ dendritic cells dritic cell differentiation (n=3). The anti-proliferative and we found no evidence for a differential capacity capacity of MSC was analyzed by co-culturing CSFE- between the different subpopulations. labeled peripheral blood mononuclear cells with the indi- cated MSC subsets and stimulating the mononuclear cell Single cell analysis of MSCA-1+CD56± clones proliferation either with interleukin-2 and OKT3 or with The growth characteristics, phenotype, and develop-

A

CD56+ C1

CD56+ C2

CD56− C12 Foundation CD56− C31

CD105 CD166 CD90 CD73 CD34 CD45 CD271 CD56 W8B2 FZD9 Storti B

CD56+ C1 CD56+ C2

©Ferrata Figure 5. Phenotype and differentiation capacity of mesenchymal stem cells derived from single cells. (A) Bone mar- CD56- C12 CD56- C13 row cells triple-stained with CD56-FITC, MSCA-1-PE and CD271-APC were gated and sorted as described in Figure 2C. Single cells were sorted in 96-well plates and cultured for 12 days. The

Oil Red O resulting mesenchymal stem cells colonies were transferred to T-25 flasks and cultured for 12 more days and stained with the indicated antibodies. ALP Note the heterogeneous expression pro- C files. (B) Morphology of MSCA-1+CD56+ derived mesenchymal stem cells clones C1 and C2, and MSCA-1+CD56– derived clones C12 and C13. (C) Osteogenic, Neural

β -tub.-III adipogenic, and neuronal differentiation potential of mesenchymal stem cells derived from single MSCA-1+CD56– and + +

NC MSCA-1 CD56 bone marrow cells. Note that the MSCA-1+CD56– clone C13 gives rise to adipocytes but not osteoblasts. CD56+ C1 CD56+ C2 CD56- C12 CD56- C13

| 180 | haematologica | 2009; 94(2) Novel MSC markers

Table 1. Phenotype and proliferation of mesenchymal stem cells clones (C1-C16) derived from single MSCA-1+CD56– and MSCA-1+CD56+ bone marrow cells. Clone identity CD105 CD166 CD90 CD73 CD34 CD45 CD271 CD56 W8B2 FZD9 Cell number 24 days after culture CD56+ (C1) G11 ++ + +++ ++ − − + (+) − + 450×103 CD56+ (C2) F7 ++ + +++ +++ −−+(+) + (+) 450×103 CD56+ (C3) B6 ++ ++ +++ +++ −−+++−− 75×103 CD56+ (C4) C4 ++ ++ +++ +++ + − ++− (+) 12×103 CD56+ (C5) C5 + + +++ ++ −−++−− 12×103 CD56+ (C6) B12 ++ ++ +++ +++ −−++−− 1×103 CD56+ (C7) F10 ++ ++ +++ ++ − − + + − − 3×103 CD56+ (C8) C1 ++ ++ +++ +++ −−++−− 7×103 CD56+ (C9) C6 + + +++ +++ −−++−− 9×103 CD56+ (C10) F6 ++ ++ +++ +++ + − ++−− 5×103 CD56+ (C11) B7 ++ ++ +++ +++ (+) − ++− +4×103 CD56– (C12) E9 ++ + +++ ++ + − (+) (+) (+) ++ 100×103 CD56– (C13) C4 ++ + +++ +++ −−(+) − + + 100×103 CD56– (C14) A9 ++ + +++ +++ (+) − + (+) + (+) 50×103 CD56– (C15) E3 ++ + +++ +++ (+) − (+) + − (+) 8×103 CD56– (C16) H9 ++ + +++ ++ + −− −++ (+) 6×103

+++ indicates > 100 D mean fluorescence intensity (MFI); ++ indicates 10–100 D MFI; + indicates 3–10 D MFI; (+) indicates 1–3 D MFI; – indicates negative staining.

mental capacity of individual MSCA-1+CD56+ and geneity among individual clones with regard to prolifer- MSCA-1+CD56– cells were determined by sorting single ationFoundation potential was observed (Table 1). Although the cells into gelatine-coated 96-well culture plates and cul- average number of cells derived from CD56+ clones was turing them in serum replacement medium until macro- about double that from CD56– clones (93.5×103 versus scopically visible colonies (>20 cells) appeared. 52.8×103 cells after 24 days of culture), no correlation The cloning efficiency of sorted MSCA-1+CD56+ between individual clones or phenotype profiles could and MSCA-1+CD56– cells was 11/96 and 5/96,Storti respec- be detected (Table 1). tively (clone numbers represent the mean of two indi- The highly proliferating clones C1 and C2 (MSCA- vidual experiments). This approximately 2-fold 1+CD56+) and C12 and C13 (MSCA-1+CD56–) were also increased frequency of CD56+ cells is in line with the analyzed for their osteoblast, adipocyte and neuronal 2-fold higher colony scores of bulk sorted cells differentiation potential. Figure 5C illustrates that – in described in Figure 2D. line with the results of bulk-sorted cells – only one The resulting colonies were transferred into T-25 CD56– clone but none of the CD56+ clones gave rise to flasks and expanded until they reached about 18 cell oil red O dye-incorporating adipocytes. Alkaline phos- divisions (corresponding to about 2.6×105 cells and 60- phatase-positive osteoblasts and neuronal β-tubulin-III 70% confluence). Phenotype analysis showed that all 16 expressing neuron-like cells were generated by three of clones were negative ©Ferratafor CD45 but expressed CD73, four clones but not by the CD56– clone C13 (Figure 5C). CD90, CD105, and CD166 and showed reduced CD271 Of note, β-tubulin-III-positive cells were 5- to 10-times expression (Figure 5A, Table 1). All CD56+ clones, more frequent in the CD56+ clones than in the positive except clone C3, downregulated CD56 expression. In CD56– clone. Together, these results underline the high contrast, CD56 expression was induced in two of four degree of heterogeneity of individual clones derived MSC clones derived from CD56– cells (C14, C15). from MSCA-1+CD56+ and MSCA-1+CD56– cells. Interestingly, significant CD34 expression was observed in two of each CD56+ and CD56– clones, whereas MSCA-1 was detected only in one CD56+ Discussion clone and in four out of five CD56– clones. Similarly, frizzled-9 expression was observed in only four out of We have previously identified novel candidate MSC 11 CD56+ clones and in all CD56– clones. These data subsets in bone marrow using proprietary monoclon- demonstrate that each single clone has an individual al antibodies.26 The aim of this study was the charac- expression profile with preferential expression of terization of MSC populations recognized by the MSCA-1 and frizzled-9 in CD56– clones. CD56-specific 39D5 and MSCA-1-specific W8B2 Expansion of all 16 individual cells for 24 days result- monoclonal antibodies. Phenotypic analysis showed ed in the appearance of spindle-shaped cells with that the targets of these reagents are selectively fibroblast-like morphology (Figure 5B). A strong hetero- expressed on CD271bright but no other bone marrow

haematologica | 2009; 94(2) | 181 | V.L. Battula et al.

cells. As CFU-F are only found in CD271bright cells, it adipogenic, but only 30% chondrogenic differentia- was likely that the reagents detected MSC. Indeed, tion.4 In our studies we demonstrated that each indi- CD271brightCD56+ and CD271bright MACA-1+ cells did not vidual colony of 16 tested clones displayed discrete only co-express the established MSC markers CD13, expression, proliferation, and differentiation features. CD73, CD105, and CD140b and lacked expression of Of these, only the large colonies (occurring more fre- CD133 and CD45, but they also gave rise to CFU-F. quently in the MSCA-1+CD56+ fraction) could be Interestingly, the cloning efficiency was 2 to 4 times expanded and used for differentiation assays, indicat- higher in the MSCA-1+CD56+ subset than in the ing that only these colonies are derived from immature MSCA-1+CD56– subset. In addition, effective chondro- MSC. Adipocyte differentiation could be detected cyte and pancreatic-like islet differentiation could be only in colonies derived from MSCA-1+CD56− but not induced only in cells of the MSCA-1+CD56+ fraction. MSCA-1+CD56+ cells which is in line with the results In contrast, adipocytes emerged only from MSCA- obtained from bulk-sorted cells. Although cartilage 1+CD56– cells. Sorting of single cells from both subsets differentiation of single cells was not analyzed, we not only confirmed the distinct proliferation and dif- anticipate a several log decade enrichment of chondro- ferentiation capacities of MSCA-1+CD56+ and MSCA- cyte progenitors in the MSCA-1+CD56+subset because 1+CD56– cells but also showed an unexpected degree of their almost exclusive presence in this population of heterogeneity among single clones. In fact, each and because only 1 in 104-105 bone marrow cells clone displayed a unique phenotype, proliferation rate, express this phenotype. Together, our studies confirm and differentiation capacity. the vast heterogeneity of individual MSC clones Recently, Prockop et al. described two morphologi- detected by other groups and show, for the first time, cally distinct subsets in cultured MSC consisting of that even narrowly defined, rare MSC populations are either small and rapidly self-renewing (RS) cells or of highly heterogeneous with respect to their phenotype, larger, more mature and slowly replicating cells their proliferation capacity, and their differentiation (mMSC). RS cells gave rise to low forward and side potential. scatter (FSlo/SSlo) and mMSC18,41-43 to large forward and In the past, several surface markers were identified side scatter (FShi/SShi) signals. No suitable surface mark- for the prospective isolation of bone marrow-derived er was identified to separate these subsets. When com- MSC. These markers include CD73, CD105, CD271, paring light scatter-defined, culture-derived RS cells STRO-1, SSEA-4, and GD2.4,19-26,44 However, none of and mMSC with the surface marker-defined cell sub- these markers stringently recognizes CD271brightCD45-/lo sets described in this report, a limited analogy cells. InFoundation this study, we described two antibodies, between primary MSCA-1+CD56+ cells and RS cells W8B2 and 39D5, which fulfil these criteria. Moreover, and between MSCA-1+CD56– cells and mMSC can be the CD56-specific 39D5 monoclonal antibody selec- drawn. Thus, MSCA-1+CD56+ cells reside in the FSlo- tively reacts only with a rare MSC subset.26 Although medium/SSlo and MSCA-1+CD56− cells in the more hetero- CD56 is also expressed on natural killer cells and acti- geneous FSlo-hi/SSlo-hi population (data not shown), Stortisug- vated T cells, the 39D5 epitope is surprisingly absent gesting that MSCA-1+CD56+ cells are more mature. In on these cells. There is no explanation for the differen- fact, the number and the average CFU-F size of tial expression of CD56 epitopes because all known MSCA-1+CD56+ cells was 2-4 fold increased compared isoforms vary in amino acid sequences of their intra- to MSCA-1+CD56– cells. In addition, only MSCA- cellular domain. At present, one might speculate that 1+CD56+ cells were able to effectively differentiate into the 39D5 epitope on natural killer cells is inaccessible chondrocytes, as revealed by the increased cartilage due to structural changes in this molecule during mem- pellet size and extensive staining. In con- brane assembly, to lipid compositions that inhibit the trast to RS cells, MSCA-1+CD56+ cells were unable to access of the 39D5 antibody, or to hitherto unknown differentiate into adipocytes. Instead, this capacity post-translational modifications of CD56. Further was detected in MSCA-1©Ferrata+CD56− cells, indicating that investigations are necessary to study this issue. this subset contains lineage-restricted progenitor cells, Injuries of articular cartilage and spinal disks are too. In conclusion, RS cells represent rapidly proliferat- major clinical problems because of the limited self- ing immature progenitor cells with multilineage differ- regenerating ability of this tissue. Diseases including entiation capacity, whereas MSCA-1+CD56+ cells are rheumatoid arthritis, trauma, acute osteochondral enriched for immature MSC with increased prolifera- fractures, and spinal disk injuries are directly related to tion rates but with a more restricted differentiation the lack of effective chondrogenesis.45 Despite the capacity. In contrast to MSCA-1+CD56+ cells, the progress in orthopedic surgery and increasing success MSCA-1+CD56− subset is enriched for more mature in autologous chondrocyte transplantation,46,47 cell and less clonogenic MSC but – unlike mMSC – retains biology-related approaches for cartilage regeneration multipotential cells including adipogenic progenitor remain challenging. A major concern is the use of cul- cells. tured cells for clinical purposes, the initiating cells of Several groups have reported that MSC derived from which are only poorly characterized. As an attractive single cells are heterogeneous with respect to their alternative, highly enriched and well-defined proliferation and differentiation capacity.4,18 The fre- CD271brightMSCA-1+CD56+ bone marrow cells with quency of individual clones with specific differentia- potent chondrogenic differentiation capacity may be tion potentials varied significantly. In one study, employed as the starting population. These cells may almost 100% of colonies underwent osteogenic, 80% be either used directly for injections into spinal disk

| 182 | haematologica | 2009; 94(2) Novel MSC markers spaces or expanded and differentiated in vitro into chondrocytes prior to use in clinical settings. Authorship and Disclosures In conclusion, we prospectively identified for the first time two phenotypically distinct MSC subpopu- All authors meet the criteria for being contributing lations in bone marrow with differential clonogenic authors. VLB and ST contributed to the conception and and differentiation capacity. We demonstrated that design of the study, data analysis and interpretation, the 39D5 epitope of CD56 is selectively expressed on and manuscript writing; PMB, FG and HR participated cells of a minor MSC population and that 39D5+ cells in the conception and design of the study, data analysis show increased clonogenic and proliferative potential and interpretation; PdZ and BS participated in provision as well as a unique chondrocyte and pancreatic differ- of study material; IM and TS contributed to data analy- entiation capacity. We also introduced MSCA-1 as sis and interpretation; WEF participated in data analysis novel and selective pan-MSC marker and showed that and interpretation; LK contributed to the final approval only MSCA-1+CD56– but not MSCA-1+CD56+ MSC of the manuscript; HJB contributed to the conception were able to differentiate into adipocytes. MSCA- and design of the study, data analysis and interpreta- 1+CD56+ MSC may be used as the starting population tion, and final approval of manuscript. All authors were of choice for the treatment of several diseases, in par- involved in the discussion and interpretation of data and ticular for rheumatoid arthritis, trauma, acute osteo- all approved the final version. chondral fractures, and spinal disk injuries. The authors reported no potential conficts of interest.

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Storti

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