Primary Mesenchymal Stem and Progenitor Cells from Bone Marrow Lack Expression of CD44 Protein Hong Qian, Katarina Le Blanc and Mikael Sigvardsson Linköping University Post Print N.B.: When citing this work, cite the original article. Original Publication: Hong Qian, Katarina Le Blanc and Mikael Sigvardsson, Primary Mesenchymal Stem and Progenitor Cells from Bone Marrow Lack Expression of CD44 Protein, 2012, Journal of Biological Chemistry, (287), 31, 25795-25807. http://dx.doi.org/10.1074/jbc.M112.339622 Copyright: American Society for Biochemistry and Molecular Biology http://www.asbmb.org/ Postprint available at: Linköping University Electronic Press http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-80787 Primary Mesenchymal Stem and Progenitor Cells from Bone Marrow Lack Expression of CD44 Hong Qian1,2*, Katarina Le Blanc2, Mikael Sigvardsson1 1Department of Clinical and Experimental Medicine, Linköping University, SE-58185 Linköping, Sweden. 2Center for Hematology and Regenerative Medicine, Department of Medicine, Karolinska University Hospital Huddinge, S-141 86, Stockholm, Sweden. Running title: Native mesenchymal stem cells do not express CD44 *To whom correspondence should be addressed: Hong Qian, HERM, Novum Floor 4, Karolinska University Hospital Huddinge, Hälsovagen 7, S-141 86, Stockholm, Sweden. E-mail: [email protected], Tel: +46-8-58583623, Fax: +46- 8-585 836 05. Key words: Mesenchymal stem cells, CD44, microarray, Bone Marrow, Flow cytometry. Background: Natural phenotype of mesenchymal stem cells (MSCs) has not been well- characterized. Results: MSCs from bone marrow naturally are CD44-, however, in vitro cultivation results in acquisition of CD44 expression on the cells. Conclusion: Native MSCs in bone marrow lack CD44 expression. Significance: Our findings highlight natural phenotype of MSCs and open new possibilities for prospective isolation of MSCs from bone marrow. 1 SUMMARY the information has been obtained from in vitro studies on culture-expanded cells, which Despite significant progress in our may not represent the phenotype of MSCs in understanding of mesenchymal stem cell vivo (2-5). Multi-color fluorescence activated (MSC) biology during the last years, much of cell sorting (FACS) have been fundamental for the information is based on experiments using definition and prospective isolation of in vitro culture-selected stromal progenitor different cell populations of the hematopoietic cells. Therefore, the natural cellular identity of system over the last 20 years. Recent MSCs remains poorly defined. Numerous development of FACS-based protocols for the studies have reported that CD44 expression is isolation and characterization of MSCs one of the characteristics of MSCs in both directly from BM opens the possibility to human and mice, however, we here have better identify and characterize non- prospectively isolated bone marrow stromal hematopoietic cell compartments in the BM. cell subsets from both human and mouse bone In mice, platelet derived growth factor marrow by flow cytometry and characterized receptor α (PDGFRα), stem cell antigen-1 them by gene expression analysis and function (SCA1), CD51 and Nestin are expressed on assays. Our data provide functional and freshly isolated BM stromal cell populations molecular evidence suggesting that primary enriched with MSCs (6-8). In human, several mesenchymal stem and progenitor cells of - surface proteins including Stro-1, CD271 and bone marrow reside in the CD44 cell fraction CD146 may be used as markers for in both mice and humans. The finding that - mesenchymal stem and progenitor cells (9-14). these CD44 cells acquire CD44 expression In addition, expression of markers such as after in vitro culture provides an explanation to CD105, CD90 and CD49A have been the previous misconceptions concerning CD44 diversely reported to be characteristic of expression on MSCs. In addition, the other MSCs (15). Among those, CD44 has been previous reported MSC markers including reported to be highly expressed on in vitro CD73, CD146, CD271, CD106/VCAM1 are expanded MSCs from both humans and mice also differentially expressed on those two cell (16-22). CD44 is an adhesion molecule types. Our microarray data revealed distinct - existing in different isoforms that interact with gene expression profile of the freshly CD44 multiple ligands such as hyaluronan, selectins, cells and the cultured MSCs generated from collagen and fibronectin (23). It is widely these cells. Thus, we conclude that bone expressed in multiple cell types including marrow MSCs physiologically lack expression hematopoietic cells, cancer stem cells (24). of CD44, highlighting the natural phenotype of MSCs and open new possibilities to In the present study, by using multi- prospectively isolate MSCs from the bone color FACS, microarray analysis and CFU-F marrow. assay, we have found that while freshly isolated MSCs from human and mouse BM express the surface markers previously INTRODUCTION reported to mark early mesenchymal Mesenchymal stem cells (MSCs) was progenitors, they lack expression of CD44. originally isolated from bone marrow (BM) by Further characterization of the cells revealed their capacity to generate colony-forming unit- that the CD44+ cells displayed little or no fibroblast (CFU-F) in vitro (1). Although there CFU-F activity whereas the CD44- cells has been significant progress in understanding contain almost all the clonogenic cells with of the biological features of MSCs, much of multilineage differentiation potentials. 2 However, in vitro culture of the CD44- MSCs excluded by propidium iodide (PI) staining. and progenitor cells resulted in their The cells were analyzed and sorted on conversion to a CD44 positive phenotype, FACSAria II Sorp (BD). providing an explanation to the previous observations suggesting CD44 as a marker for FACS isolation and analysis of mouse MSCs. MSCs. Furthermore, the cultured MSCs The BM mononuclear cells from femurs, tibias derived from the fresh CD44- stromal cells and iliac crest of FVB/N mice were isolated display distinct gene expression profiles of cell using a standard protocol which was tested in adhesion molecules and growth factors as well our laboratory without affecting cell surface as cytokines. These findings highlight the marker expression. The bones were first importance of in vivo/ex vivo analysis of crushed in PBS+10%FBS (PAA) in order to mesenchymal cells for identifying their obtain maximal cells in BM endosteal region physiological properties and suggest that prior to enzyme treatment. The marrow cells CD44 expression can be used as a negative were collected and the bone fragments were rather than a positive marker for prospective then treated with 0.1% Collagenase II (CLS II isolation of MSCs from BM. Worthin gton Biochemical) and 0.05% trypsin-EDTA for 45 min at 37°C. The tubes EXPERIMENTAL PROCEDURES were shaken every 10min during incubation. Subject: BM aspirates were obtained from The treatment was stopped by adding ice cold iliac crest of normal young adult volunteers FBS to reach a final concentration of 20% following informed consent according to the FBS, subsequently wash the bones by procedures approved by local ethics committee PBS+10% FBS. The cells were collected and at Karolinska Institute (Stockholm, Sweden). filtered via 70 µm cell strainer (BD). The bone Mouse bones were obtained from adult (3-4 and marrow cells were pooled and spun down month old) normal FVB/N mice. Animal at 300g for 10 min and then resuspended in procedures were performed with approval PBS+10% FBS. The stromal cells were first from the ethics committee at Linköping enriched by depleting hematopoietic cells University (Linköping, Sweden). using purified rat anti-mouse antibodies against CD45 and LIN (TER119, B220, CD4, FACS isolation and analysis of human BM CD8, GR1 and MAC1) and subsequently MSCs. Mononuclear cells from BM aspirates using sheep anti-rat Dynal beads (Invitrogen). of healthy adult volunteers were isolated by The endothelial cells and the residual Ficoll-Hypaque (Lymphoprep, Axis-Shield hematopoietic cells were visualized by CD31 PoC AS) density centrifugation. The CD45- - and goat-anti-rat tricolor antibody and/or CD235 cells were enriched by negative CD45 and TER119. The dead cells were selection using CD45 and CD235 microbeads excluded by PI staining. The CD44+ and and magnetic-activated cell sorting (MACS, CD44- stromal cells were gated or analyzed Miltenyi Biotec). The cells were then stained based on fluorescent minus one (FMO) with anti-human CD271 CD146, CD105, controls for CD44 expression on FACS aria II CD106, CD73, STRO-1, CD29, CD45 and Sorp (BD). For information about the Glycophorin A/CD235. Anti-human CD19 antibodies used in the study, see Supplemental was included in the staining in order to information. exclude possible contamination of B cells in the sorted stromal cells. For information about the antibodies used in the study, see CFU-F assay. The stromal cells (CD45-LIN- supplemental information. Dead cells were CD31-CD44+ and CD45-LIN- CD31-CD44-) 3 from normal mouse and human BM were Assays-on-Demand probes, see below list of sorted and plated into 96-well plates or 12- probes used for Q-PCR. well plates containing complete Mesencult medium in MesenCult® Proliferation Kit for Cell Cycle Analysis. The analysis was mouse (#05511) and human (#05411), performed as described(25). BM mononuclear respectively (Stem cell Technologies, cells from wild-type FVB/N mice were Vancouver, Canada) in hypoxic (1% O2) for initially stained with antibodies against CD45, 10-12 days (mouse) or 12-14 days (human). lineage cells and CD44. After incubation with The mouse cells were seeded at a density of the cell surface antibodies, the cells underwent 10, 40, 100 and 200 cells/well for the CD44- fixation with a Cytofix/Cytoperm kit (BD cells and 100, 200, 400, 1000 and 2000 Biosciences) and staining of PE-anti-KI67 and cells/well and the human cells were plated at DAPI. Analysis was performed on a FACS 2, 5, 10 cells/well for the CD44- cells and 10, ARIA II SORP (BD Biosciences). 50, 100 and 1000 cells/well for the CD44+ cells.