Jacobs Journal of Regenerative Medicine

OPEN ACCESS Jacobs Journal of Regenerative Medicine

Research Article Isolation and Characterization of Early Lineage Adult Stem Cells from the Synovial Fluid of Osteoarthritis Patients Keith D. Crawford1, 2, *, Baldev Vasir3, Shari Benson1, Jinsoo Joo1, Kathryn A. Goldman1, Zaheed Husain4, Farnaz Hadaegh5, Thomas S. Thornhill6 1Center for Molecular Orthopedics, Department of Orthopedic Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, 02115, USA 2Asclepius Laboratories, Inc, 27 Strathmore Road, Natick, MA, 01760, USA 3Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, 02215, USA 4Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, 02115, USA 5Department of Anaesthesia, Massachussets General Hospital, Harvard Medical School, Boston, MA, 02114, USA 6Department of Orthopedic Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, 02115, USA *Corresponding author: Dr. Keith D. Crawford M.D., Ph.D., Asclepius Laboratories, 27 Strathmore Road, Natick, MA 01760, USA, Tel: (202) 538-3336; Fax: (314) 222.6604; Email: [email protected] Received: 03-14-2016 Accepted: 03-28-2016 Published: 03-31-2016 Copyright: © 2016 Crawford

Abstract

Adult stem cells (ASCs), which possess the ability to self-renew and regenerate tissue, are of significant value for the develop methodologiesment of cellular and therapies, used a tissuedifferent engineering approach tools, to identify and drug early screening lineage adultmodels. (ELA) Conventional stem cells, protocols a subpopulation for ASC enrichmentof ASCs 4-6 generate a small number of cells that do not represent the total ASC population of tissues. We avoided these conventional

µm in diameter, in the synovial fluid of osteoarthritic patients. These cells lack cell surface markers expressed by other ASC (e.g. CD34, CD73, CD105, SSEA-1, CXCR4). However, RT-PCR studies demonstrated expression of pluripotency genes such as NANOG, OCT4, REX1, KLF4, STELLA, and SOX. When cultured in adipogenic, chondrogenic, or osteogenic differentiation media, ELA cells differentiated into fat, cartilage, and bone tissue, respectively. Quantitative PCR analysis revealed unique molecular signatures consisting of tissue-specific and non-tissue specific mRNA in the differentiated tissues, suggesting a- continuum of mRNA expression. Furthermore, the ELA cell population shared unique gene sets with embryonic stem cells, mesenchymal stem cells, and induced pluripotent stem cells. Some of these genes are unique to neuronal, cardiac, pancreat ic, and hepatic progenitor cells, while others, such as mucins, ICAM, and tetraspans, have tissue-specific cell functions. ELA cells also demonstrated strong in vitro immunomodulatory properties by inhibiting T cell proliferation, inducing CD4+/ CD25+ regulatory T cells, and inhibiting natural killer cell activity. Collectively, these observations suggest that ELA cells Keywords:might be useful for cell-based regenerative therapies and the treatment of systemic diseases with immunological etiologies.-

Osteoarthritis; Synovial fluid; Bone marrow; Adult Stem Cells (ASCs); Self-Renewal; Heterogeneity; Multipo Introductiontent, Differentiation, Transcriptome, Early Iineage Adult (ELA) Stem Cells; Molecular Signatures - poietic stem cells (HSCs), one of the most characterized types fetus during tissue renewal and postnatally [6,7]. Hemato - ferentiate into multiple cell lineages, and reconstitute tissue Stem cells have the remarkable capacity to self-renew, dif of ASCs, have been studied for over 50 years and are known progenitors of various blood cell types [8-10]. HSCs have type, are established from early embryonic cells and possess in vivo [1]. Embryonic stem cells (ESCs), a pluripotent cell isbeen also used a heterogeneousclinically to reconstitute population bone of non-hematopoieticmarrow (BM) cells destroyed by BM ablation therapy for cancer [11,12]. There- the ability to differentiate into all three germ layers [2-5]. In stem cells in the BM. In particular, mesenchymal stem/pro contrast,Cite this article: adult Crawford stem K D. cells Isolation (ASCs) and Characterization are found in of Earlythe developinglineage Adult Stem Cells from the Synovial Fluid of Osteoarthritis Patients. J J Regener Med. 2016, 2(1): 005. Jacobs Publishers 2 aregenitor found cells in (MSCs)the peripheral are also blood,thought umbilical to originate cord from blood, the adiBM- sponse, potentially by inhibiting T cell proliferation, inducing and comprise 0.01-0.001% of nucleated BM cells [13]. MSCs regulatory CD4+/CD25+ T cells, and inhibiting natural killer- itantly(NK) cell with activity. MSCs, Thus, to allow like newMSCs, therapies ELA cells for might a wide participate range of pose tissue, skeletal muscle, liver, lungs, synovium, dental pulp, commonin various and regenerative orphan diseases processes, or they might work concom apical papilla, amniotic fluid, and fetal blood [14]. Because MSCs are found in extremely low numbers in the BM, sustained Materials and Methods [23]. ex vivo culture on tissue culture plastic is required to generate sufficient cell numbers for phenotypic characterization [15]. MSCs most commonly express surface markers such as CD29, SF isolation and cell culture CD44, CD49a-f, CD51, CD73, CD105, CD106, CD166, and Stro1- and lack expression of hematopoietic lineage markers such as - CD11b, CD14, and CD45 [16]. MSCs are multipotent ASCs capa thatble of MSCs differentiating are capable into of variousdifferentiating mesodermal into ectodermal tissues, such and as SF was extracted from the knees of patients diagnosed with os- adipose, cartilage, and bone [16]. Other groups have reported- teoarthritis (OA) following appropriate institutional approved protocols. Within 24 h of harvest, the SF was diluted 10:1 in di repairendodermal tissue tissues,but instead such secrete as lung, trophic skin, pancreas, factors that and decrease liver tis lution buffer (phosphate-buffered saline (PBS) supplemented cellsue death,[17]. It recruit has been immune hypothesized cells to the that injury MSCs site, may and not promote directly with 10% fetal bovine serum (FBS; HyClone, Logan, UT)) and 10 mM ethylenediaminetetraacetic acid (EDTA; Gibco, Grand- Island, NY). To extract the cellular component, the diluted SF healing [18,19]. Presently, MSC immunomodulatory properties was spun at 500 x g for 30 min, and the pellet was resuspend are being assessed in clinical trials to determine their efficacy ed in dilution buffer. This process was repeated twice at 300 in treating a variety of immune-related diseases [20]. x g for 30 min, and the final pellet was resuspended in Hank’s balanced salt solution (HBSS; Gibco). The pelleted cells were either directly analyzed or subjected to culture expansion and Most ASC studies focus on BM-derived stem cells and use differentiation were suspended in growth medium (MSCGM™ differentiation. Samples that underwent culture expansion and discontinuous density gradients, such as Ficoll-Paque and- Lymphoprep, and plastic adherence to enrich for ASCs [21]. - Human Mesenchymal Stem Cell Growth BulletKit™ Medium or Although density gradients effectively separate debris, plate MSCGM-CD™ Mesenchymal Stem Cell Chemically2 Defined Me lets, and red blood cells (RBCs) from the mononuclear cells in plated at a concentration of 3000 cells per cm (225,000 cells dium with or without 1% FBS; Lonza, Basel, Switzerland) and wethe buffy decided layer, to they forgo also the inadvertently use of discontinuous discard a subset gradients of ASCs and [22]. To avoid potential discrepancies in the isolation of ASCs, Jose, CA). All culture media were total) in a T-75 vented tissue culture flask (BD Biosciences, San - supplemented with 100 U/ prolonged culture on tissue culture plastic to harvest ASCs. We - ml penicillin and 1000 U/ml streptomycin (PCN-Strep; Gibco) chose synovial fluid (SF) as a source due to low RBC contami in and exchanged every 48 h. Once cells reachedoC >80% with 5%conflu CO nation. To isolate ASCs, we used time sedimentation of diluted 2 diameter (mean = 5 ence, they were harvested with Trypsin-EDTA and replated SF. Cells in the enriched ASC population measured 4-6μm into new T-75 flasks. Cells were cultured at 37 μm). Flow cytometry and gene expression and proteins generally thought to be restricted to ESCs. In ad- for all experiments. Samples were either immediately seeded analysis suggested that this ASC population expresses genes into cell culture or mixed 1:1 with freezing medium composed of Dulbecco’s Modified Eagle’s Medium (Gibco) supplemented dition, these cells did not express MHC class II, CD44, CD45 with 20% FBS and 20% dimethylsulfoxide (Sigma-Aldrich) or CD49, but unlike our prior studies they were smaller and and stored in liquid nitrogen at less than -150°C. had minimal MHC class I expression. Semi-quantitative PCR- Flow cytometric characterization of ELA cells cy.studies We thereforeshowed expression named these of embryonic cells early transcription lineage adult factors (ELA) such as OCT4, REX1, NANOG and SOX2, suggesting pluripoten - the cells described in earlier studies. stem cells, since they shared the gene expression profiles of Antibody use was based on the minimal surface marker pan - el proposed by the International Society of Cellular Therapy [16]. Fluorochrome-labeled antibodies against the following In this study, we investigated the ability of ELA cells to self-re markers and matching isotype controls were obtained from new and differentiate into multiple lineages. Moreover, we BD Biosciences: CD44-PE, CD45-PE, CD49-PE, CD105-PE, intooptimized adipose, the cartilage, isolation, and culture, bone and lineages, expansion and conditionsthat they also for CD133-PE, CD34-PE (clone 581), CD73-PE, CD90-PE, CD99-PE, these cells in vitro. We show that ELA cells can differentiate- CD235a-PE, MUC1-PE, HLA Class1-PE, HLA-DR-PE, SSEA1-PE, mined that ELA cells are potent modulators of the immune re- HLA-DR-PE, IgG1-PE, IgG1-FITC, IgG2a-PE, IgG2bkappa-PE, express genes from other cell types. In addition, we deter IgM-PE, CD4-FITC, CD8-FITC, CD69-PE, CD3-FITC, and CD25- PE. Anti-CXCR4-PE (CD184) antibody was obtained from R&D Jacobs Publishers 3 - - Systems (Minneapolis, MN). Anti-PD1-PE2 (CD279) antibody Medium (Invitrogen) with Chondrogenesis Supplement (Invit was obtained from eBiosciences (San Diego, CA). Upon con- rogen). These cell cultures were then stained with Alcian Blue fluence, cells from one 75 cm flask were harvested, washed,- (Sigma-Aldrich) for chondrocyte detection. For osteogenic trifugationand counted. and Cells aspiration were kept of onsupernatant, ice and suspended 5 or 10 in incubaof anti- differentiation, cells were cultured in Osteocyte/Chondrocyte- bodytion buffer (depending (Dibco’s on PBS cell + number) 2% FBS + was 1 mM applied EDTA). directly After ontocen blasts,Differentiation cell cultures Basal wereMedium stained (Invitrogen) with 5-bromo-4-chloro-3- with Osteogenesis μl Supplement (Invitrogen). To assess the presence of osteo- resuspended, and analyzed the pellet.™ Cells were incubated at 4°C for 30-45 min, washed,- indolyl-phosphate/nitro blue tetrazolium (NBT/BCIP; Invit in a FACSCalibur machine using phaserogen). microscopy. In all three differentiation studies, positive cells were intracellularCellQuest software staining (BDusing Biosciences). monoclonal antibodiesThe pluripotent against prop OC- assayed by counting 50-100 cells in multiple fields using light erties and ESC marker status of ELA cells were determined by RNA isolation and RT-PCR TA4-PE, RUNX2-PE, SOX9-PE, REX1-PE, NANOG-PE, and KLF4- PE with matching isotype controls and by RT-PCR of freshly - isolated and culture-expanded cells. For intracellular staining, panded undifferentiated cells as well as differentiated cells. Total RNA was extracted from freshly isolated and culture-ex withcells werethe appropriate permeabilized antibodies. with Cytofix/Cytoperm solution (BD Biosciences) and thereafter subjected to intracellular staining Total RNA was purified using TRIzol® reagent according to Self-renewal properties of ELA cells below).the manufacturer’s protocol (Invitrogen). The same source of- RNA was used for RT-PCR and DNA microarray analysis (see- First-strand cDNA was obtained by reverse transcrip tion using 3 mg total RNA according to the manufacturer’s in ELA cell cultures grew as monolayers. Following cell sorting, structions (Invitrogen). Primer sequences are shown in Table both marker-positive and marker negative cultures were set was1. PCR synthesized products were using electrophoresed the SuperScript on 1.5% agarose gels to- up in parallel. At days 3 and 5, cultures were rinsed with PBS, verify DNA fragment sizes. For DNA microarrayTM analysis, cDNA anddetached 10 with trypsin-EDTA, centrifuged, and resuspended III First-Strand Syn in media. Duplicate aliquots were placed into 96-well plates, thesis System (Miltenyi Biotec, San Diego, CA). RT-PCR assays μl of Cell Counting Kit-8 solution (Dojindo Molecular were performed using qPCR Mastermix Plus for SYBR Green- Technologies Inc., Gaithersburg, MD) was added to each well.- (Miltenyi Biotec) according to the manufacturer’s protocol (see Following 3 h incubation at 37°C, A450 was measured using below). For normalization, differential levels of gene expres a Victor5 Light Luminescence Counter (PerkinElmer Life Sci sion were calculated in relation to beta actin and expressed as ences, Boston, MA) and compared with standards of known a ∆CT value, as previously described [24]. - Preparation for DNA microarray analysis cell numbers. To detect apoptotic cells, cultures were fixed and protocol.stained with the fluorescence-based ApoAlert DNA Fragmen tation Assay Kit (BD Biosciences) following the manufacturer’s - Adipogenic, chondrogenic, and osteogenic differentiation of Total RNA was extracted from synovial ELA cells in a mono cells isolated from synovial fluid layer of human synovial ELA cells cultured for 3 d. Cells were lysed using the SuperAmp preparation kit and delivered to Miltenyi Biotec on dry ice. SuperAmp RNA amplification was - performed according to Miltenyi Biotec’s protocol based on a edELA in cells 75cm were2 suspended in chemically defined media with global PCR protocol. mRNA was isolated using magnetic bead 1% FBS, passaged2 upon reaching 80% confluence, and plat andtechnology. 250 ng of Amplified each cDNA cDNA were samples used as were template quantified for Cy3 using and passages, cells vented were plated cell culture into 12-well flasks atplates a concentration at a concentra of- an ND-1000 Spectrophotometer (NanoDrop Technologies), 150,000/cm , with a total volume of 25 ml per flask. After 20- Cy5-labeled cDNAs were combined and hybridized for 17 h at 65Cy5C labeling to the Agilentaccording Whole to Miltenyi Human Biotec’sGenome protocol. Oligo Microarray Cy3- and tion of 200,000 cells/well and cultured in the appropriate differentiation media. For adipogenic differentiation, cells were ° cultured in StemPro Adipocyte Differentiation Media (Invit 4x44K probe set using Agilent’s recommended hybridization rogen) supplemented with PCN-Strep at a total volume of 1.5 chamber and oven. Control samples were labeled with Cy3 and ml/well. The media was changed every 48 h. After 21 d, the experimental samples were labeled with Cy5. Data processing and analysis cells were harvested for histochemical staining and real-time- quantitative PCR (qPCR). Differentiated cells were stained with fresh Oil Red O solution (Sigma-Aldrich) to verify adi pocyte characteristics. For chondrogenic differentiation, cells Feature Extraction Software (Agilent) was used to read and were cultured in Osteocyte/Chondrocyte Differentiation Basal Jacobs Publishers 4 - process the microarray image files and raw datasets. These ELA cells were co-cultured with T cells labeled with 5-6-car- datasets, together with publically available datasets from the boxyfluorescein diacetate succinidyl ester (CFSE; Cell Trace- inputNIH Gene datasets Expression were transformed Omnibus (GEO), into log were base exported 2, and row-by- to JMP Cell Proliferation Kit; Molecular Probes/Invitrogen Life Tech rowsoftware statistics (SAS Institute were computed. Inc., Cary, Datasets NC) for further were normalized analyses. The to nologies) in 96-well plates at 1:10, 1:20, and 1:40 ratios in trip licate, along with non-ELA cell controls. T cell proliferation was- stimulated with CD3/CD28 and analyzed with flow cytometry the median global intensity [25,26]. for CFSE fluorescence after 5 d. Immunosuppressive proper Hierarchical clustering and functional analysis - ties of MSCs (Lonza, Walkerville, MD) were assayed in parallel with the same methods. Flow cytometry data was analyzed us - ing FlowJo software (Ashland, OR) to obtain the ProliferationAs a - Index (PI). T cell suppression for each sample was calculated To identify genes expressed at high levels in ELA cells, an un- as (1- ([PI with ELA cells]/[PI without ELA cells]) x 100. supervised hierarchical clustering was performed on the nor alize this clustering. One-way ANOVA was performed on the final assay for immunosuppression, freshly isolated ELA cells- malized dataset. JMP Software was used to perform and visu and PBMCs were co-cultured 1:10 in 96-well plates for 5-7 plot was generated to represent the intensity ratio for each days, harvested into 5 ml tubes, and labeled with a combina data obtained from the hierarchical clustering, and a volcano ratio 2 tion of directly conjugated antibodies as follows: CD4-FITC/- of the gene intensities. A log2 - CD25-PE; CD8-FITC/CD25-PE; CD3-FITC/CD69-PE, and CD3- gene in ELA cells and MSCs. The x-axis displays the log 10 FITC/PD1-PE, as well as matching isotype controls. The per- for the comparison between ratioELA cellsof 1corresponds and MSCs. Genes to approx that centages of CD4+ or CD8+ T cells expressing CD25, and CD3+ imately two-fold change. The y-axis shows the –log (p-value)- T cells expressing CD69 or PD1 were determined by bi-dimen sional FACS analysis. were differentially expressed in ELA cells and MSCs were iden program.tified. These genes, along with their fold-change values, served with ELA cell suppression of NK cell activity was determined by a as the input to the Ingenuity Pathway Analysis (IPA®, Qiagen)- Differently expressed genes were uploaded into the (Mediatech,chromium release Herndon, assay VA) [29]. supplemented NK cells were with co-cultured 10% pooled hu- equal numbers of ELA cells in RPMI 1640 tissue culture media IPA=0.05 application and the whole and used database as the as startinga reference point set forwas generat used to determineing biological if the networks enrichment [27]. of A genes right-tailed with particular Fisher’s biologicaltest with α man AB serum, antibiotics, and cytokines for 24 h. Following incubation, NK cells were transferred to wells for co-culture- functions or molecular processes was significant. with chromium-labeled K562 target myeloma cells (ATCC, Immunomodulatory properties of ELA cells Manassas, VA) at 10:1, 5:1, 2.5:1, and 1:1 ratios. Specific cyto toxicity was calculated as 100 x (experimental-spontaneous)/ (maximum-spontaneous). Statistical analysis The immunosuppressive properties of ELA cells were assayed triplicate.by several ELA methods. cells were Suppression irradiated of in T suspension cell proliferation with a dose was determined by in vitro co-culture experiments performed in t Results are expressed as mean ± SEM. Statistical comparisons Irradiated and non-ir- of 30 Gy prior to coculture using the Gamma cell Elan device were performed using the Student’s -test. P values <0.05 were considered statistically significant. (Best Theratronic, Ottawa, ON, Canada). - Results radiated ELA cells, either freshly isolated or cryopreserved and then cultured for 24-48h, were co-cultured with freshly isolat Cellular phenotypes of SF cells ed human peripheral3 blood mononuclear cells (PBMCs) at a- 1:10 ratio for 5 d at 37°C. ELA cell suppressive function was- determined by a [ H]-Thymidine (1μCi/well; 37kBq; NEN-Du Pont) uptake assay, as previously described [28]. Data are ex - SF contains a wide variety of mononuclear cells, some of which- pressed3 as counts per minute (cpm) or as a stimulation3 index - gions,revealed three a forwardof which and represent side scatter neutrophils, flow cytometry myeloid cells, pattern and (SI). SI was determined by calculating the ratio of experimen similar to peripheral blood. This pattern consists of four re tal [ H]-Thymidine incorporation to background [ H]-Thymi - cells,dine incorporation which would beby unstimulatedindicated by higher T cells. counts These inexperiments a prolifer- lymphocytes (Figure 1A). The fourth region, much smaller in ationalso assessed assay. if allo-reactive T cells were stimulated by ELA size and side scatter, was previously thought to primarily rep resent cell debris and RBCs (Figure 1B). We found that this- population had less fluorescence compared with other regions and <200 forward scatter (FSC) linear units (Figure 1A). Anal To further demonstrate their immunosuppressive properties, ysis of this cell population from 5 OA patients revealed a mean Jacobs Publishers 5 viability of 94 ± 0.65% and a mean cell size of 5.9 ± 0.31 μm NAs were expressed in this small cell population, with REX1 (range: 4-8 μm) (Figure 1C). being the most highly expressed (Table 1, Figure 1E). Notably, we identified a splicing variant of NANOG, which might suggest- a greater diversity of self renewal and pluripotency proteins. The Ntera cell line was used as a positive control in these stud ies [30], and a sample lacking reverse transcripts was used as Table 1 Primer sequences for RT-‐PCR ampliﬁca8on of target genes a negativeGene control. Primer Gene Bank accession number Product size (bp) GAPDH NM_001256799.1 66 F: 5'-‐ AGCCACATCGCTCAGACAC-‐3' NANOG R: 5' -‐ GCCCAATACGACCAAATCC-‐3' NM_024865.2 88 F: 5' -‐ TGTCTTCTGCTGAGATGCCT-‐3' SOX2 R: 5' -‐ TCTCTGCAGAAGTGGGTTGT-‐3' NM_003106.3 151 F -‐ 5' -‐ AGCTCGCAGACCTACATGAA-‐3' 297 OCT4. R: 5' -‐ TGGAGTGGGAGGAAGAGGTA-‐3' NM_002701.4 F: 5' -‐ ACATGTGTAAGCTGCGGC C-‐3' 105 REX1 R: 5' -‐ GTTGTGCATAGTCGCTGCTTG-‐3' NM_020695.3 F: 5' -‐ GGATCTCCCACCTTTCCAAG-‐3' GDF3 R: 5' -‐ GCAGGTAGCACACCTCCTG-‐3' NM_020634.1 156 F: 5' -‐ TGCTGTTCACTTCAACCTGC-‐3' STELLA R: 5' -‐ AGGGAGCATCTTAGTCTGGC-‐3' NM_199286.3 (AY230136.1) 65 F: 5' -‐ GGAAGCTTTACTCCGTCGAG-‐3' KLF4 R: 5' -‐ GCCACTCATCTTCGATTTCC-‐3' NM_004235.4 139 F: 5' -‐ CGTTGACTTTGGGGTTCAGG-‐3' R: 5' -‐ GCGAACGTGGAGAAAGATGG-‐3' Figure 1. Identification of early lineage adult (ELA) stem cells in ELA cell growth and self-renewal the synovial fluid (SF) of patients with osteoarthritis (OA). (A)

Representative forward- and side-scatter profiles of mononuclear cells isolated from the SF of an OA patient indicating the location of a- In order to investigate and optimize cell growth, ELA cells were- small cell population in relation to other cell types. (B) Forward- and cultured in three different media types; standard expansion Viabilityside-scatter and profile cell-size of adetermination gated small population of a gated of small cells depictingcell population a het medium, chemically defined medium (CD), and CD supple 6 6 erogeneous population with a varied cell size and scatter profile. (C)- mented with 1% FBS. ELA cells and cultured 2.2 X 10 in standard medium Doublingreached 90% times confluence for both samplesin 8 days were for donor greater A andthan 7 120 days h. for In using the Roche CASY Cell Counter and Analyzer System. (D) Expres contrast,donor B, generatingwhen ELA cells 2.8 xwere 10 cultured in CD cells, in the respectively. absence or sion of pluripotent intracellular and surface markers, as determined- - sionby FACS in a analysis small cell of apopulation gated population isolated of from small three cells inseparate Group 1,samples Group 2, and Group 3. (E) RT-PCR analysis of pluripotency marker expres- appearedpresence of to 1% be FBS, the optimalhigher yields media were for generated self-renewal with capacity, short er doubling times. Notably, 6 CD supplemented with 1%6 (donor FBS of SF, with NTERA-2 cells (a stem cell line) as a positive control. Prim ers are specific for transcripts from the respective endogenous locus. generating yields of 31.5 x 10 (donor A) and 21.2 x 10 GAPDH was used as a loading and internal control. B) ELA cells with doubling times of 19h and 27h, respectively- gy(Figure was round2A-B). immediately We therefore after chose plating this but culture became media elongated for all cells by staining for proteins associated with peripheral blood future experiments. In CD media with 1% FBS, cell morpholo We next evaluated the phenotypic characteristics of these and spindle-like within 4 d (Figure. 2C-D). mononuclear cells and ASCs. Further flow cytometric analysis- showed three distinct subgroups of cells (Figure 1B). CD45 and CD235a, surface markers of leukocytes and RBCs, respec tively, were absent from this cell population. MHC Class I, a- protein found on all cells except RBCs and immature stem cells, was observed on a subset of this group (Figure 1D). Further- more, we observed very high expression of MUC1 and CD99 on this cell population. Further analysis revealed no expres sion of CD73, CD90, CD105, CD133, CXCR4, or SSEA-1 on any of the subgroups. However, intracellular staining with directly conjugated monoclonal antibodies revealed high expression of REX1 and varying degrees of OCT4, NANOG, SOX9, and RUNX2- Figure 2. Cell culture and self-renewal properties of early lineage expression (Figure 1D). RT-PCR analysis from three patients revealed that most of the tested pluripotency-associated mR Jacobs Publishers 6 adult (ELA) cells in vitro. cells from two separate donors cultured in standard culture media or the acid mucopolysaccharides and glycosaminoglycans of cartilage. (A) Bar graph depicting growth of ELA- drogenic differentiation was assayed with Alcain Blue, which labels- chemically defined (CD) culture media with or without 1% fetal bo Diffuse blue staining was observed throughout the slide. (C) Osteo vine serum (FBS). (B) Phase micrographs demonstrating pattern and genic differentiation was assayed with BCIP/NBT, a substrate that density of ELA cell growth at two different magnifications. (C) Phase turns purple in the presence of alkaline phosphatase. Uninduced cells micrographs exhibiting the pattern and density of ELA cell growth at- were used as negative controls in the differentiation experiments. days 1, 4, and 7 in CD media supplemented with 1% FBS from three Total RNA was extracted from these differentiated cells, and cDNA Numbersseparate donors.in parenthesis (D) Total represent ELA cell population counts from doubling three separate time during do derived from mRNA was amplified based on the global PCR protocol labelednors cultured passage in growthCD media period. with 1% FBS at different passage numbers. described in Materials and Methods. (D-F) Real time RT-PCR analysis of selected specific genes expressed in differentiated ELA cells. The- ELA cell differentiation expression of genes was compared to the expression of beta-actin as differentiatedan internal control into adipocytes.and the values (E) expressedELA cells differentiatedas ΔCT. Negative into bars chon- in dicate a decrease in expression of that particular gene. (D) ELA cells chondrogenic,The differentiation and osteogenic potential of differentiation. ELA cells was Cells investigated cultured drocytes. (F) ELA cells differentiated into osteocytes. by culturing cells under conditions that favored adipogenic, - enceIn order of adipogenic,to further investigate chondrogenic, the extent and osteogenic of induced genes ELA cell by in Adipocyte Differentiation Media for 21 d formed vacuoles differentiation, cells were harvested to investigate the pres Oilthat Red stained O. Cells positive cultured for Oil for Red 21 O,d ina fat-soluble Chondrocyte dye Differentia that stains- lipids (Figure. 3A). Control cells showed no incorporation of qPCR. Cells from the adipogenesis conditions showed high- production of acid mucopolysaccharides and glycosaminogly- expression of the adipocyte lineage genes PPARG-tv1, PPARG- tion Media exhibited diffuse Alcian Blue staining, suggesting andtv2, LPL,osteogenic FABP4, conditions. ADIPOQ, LEP, Cells PLIN, from and the CFD chondrogenic (Figure 3D). con Ad- ipogenesis-specific genes were also detected in chondrogenic cans normally found in cartilage (Figure 3B). Cells cultured in- Osteocyte Differentiation Media and stained with NBT/BCIP ditions showed high expression of the chondrocyte lineage revealed flat, purple cell bodies (Figure 3C). NBT/BCIP is con genes BGN, DCN-tvA2, ANXA6-tv2, MMP13, SRY, and COMP controlverted intocells. purple stain by alkaline phosphatase, an enzyme undergoingand low/absent adipogenesis expression or of osteogenesis. MATN1 and Cells COL2A1 from (Figure. the os- found in osteoblasts. No enzymatic activity was observed in 3E). These genes were also detected at similar levels in cells

teogenic conditions showed high expression of the osteocyte lineage genes RUNX2-tv3, RUNX2-tv1, RUNX2-tv2, and PHEX, similar to the chondrogenic conditions. RUNX2.2 and PHEX were also detected in adipogenesis conditions. Low/absent- expression of BGLAP, SPP1, SPP2, and SPP3 was also observed Althoughin the osteogenic cells were conditions cultured (Figurein specialized 3F). There media was for low/ab differentiation,sent expression the differentiation of all lineage-specific process was genes not absolute in control because cells.

tocells determine also expressed optimum genes culture from conditions other lineages for stem (Figure cell 3D-F).differentiation,These observations and that molecularsuggest that signatures detailed studiesmay be are necessary necessary to

define the differentiation process. -

Large-scale gene expression profiling of freshly isolated, un differentiated ELA cells showed expression of differentially- tentialexpressed of ELA progenitor cells to differentiate and tissue-specific into other genes lineages, with such diverse as functions (Table 2). These profiling studies suggested the po Figure 3. Differentiation of early lineage adult (ELA) cells to adipocytes, chondrocytes, and osteocytes. - neuronal, cardiac, pancreatic, and liver cells (Tables 2 and 3).- The differentiation po- Moreover, expression of genes with specific cellular functions,- ferentiation.tential of ELA (A) cells Adipogenic was investigated differentiation by culturing was indicated cells for by21 accumu d under- stead,such as ELA mucins, cells mightICAM, betetraspans, a heterogeneous and collagens population (Table of 3), multi- sug conditions that favored adipogenic, chondrogenic, or osteogenic dif- potentgests that cells ELA with cell the phenotype capacity to may differentiate not be tissue into endodermal, specific. In mesodermal, and ectodermal cells. lation of neutral lipid vacuoles that stained with Oil Red O. (B) Chon Jacobs Publishers 7

Table 2.

Selected panel of differentially expressed progenitor-‐ and tissue-‐speciDic genes from freshly-‐isolated ELA cell population

Freshly harvested ELA cells (small population) was isolated from SF of OA patient. Total mRNA from the sample was used for microarray analysis. Jacobs Publishers 8 Table 3. Expression of selected group of genes for secreted proteins (collagens/mucins) and surface-‐expressed proteins (mucins, ICAMS, and tetraspans) in freshly-‐isolated ELA cells

Gene Gene Description Chr. Location Protein Expression

COL4A1 Collagen, type IV, alpha 1 13q34 Secreted Positive expression in endothelium and in basal membranes of most epithelial tissues COL4A2 Collagen, type IV, alpha 2 13q34 Secreted Positive expression in alveolar cells, muscle and stromal cells including, in addition to the small intestine, gall bladder and parts of pancreas. COL8A1 Collagen, type VIII, alpha 1 3q12.1 Secreted High level of expression in internal elastic lamina of blood vessels and cytoplasmic and membranous locations premenopausal uterine glands. Connectie tissue, bronchus, renal glomeruli, smooth and skeletal muscles and Langerhans cells also stain positive COL9A2 1q34.2 Secreted High expression in gastrointestinal tract, renal tubules and chondrocytes and low Collagen, type IX, alpha 2 expression hepatocytes. COL9A3 Collagen, type IX, alpha 3 20q13.33 Secreted Most normal tissues show positive cytoplasmic staining. Glomerular while trophoblastic, and glial cells stain weakly or are negative. COL13A1 Collagen, type XIII, alpha 1 10q22.1 Secreted Expressed in breast, trophoblastic cells, salivary gland, seminal vesicle, urothelial cells, pancreatic ducts and squamous epithelial tissue Fallopian tube, gall bladder and bile duct cells were moderate positivity. COL14A1 Collagen, type XIV, alpha 1 8q24.12 Secreted Squamous and respiratory epithelia, hepatocytes, urinary and gall bladder, endometrium, Leydig cells gastrointestinal tract and prostate showed moderate. COL15A1 Collagen, type XV, alpha 1 9q22.33 Secreted Expressed predominantly in internal organs such as adrenal gland, pancreas and kidney and localized to basement membrane zones, functioning to adhere basement membranes to underlying connective tissue stroma. COL27A1 Collagen, type XXVII, alpha 1 9q32 Secreted Involved in the calcification of cartilage and the transition of cartilage to bone

MUC1 Mucin 1, 1q21 Cell Surface Expressed on the apical surface of epithelial cells, especially of airway passages, Associated breast and uterus. MUC2 Mucin 2 11p.15 Secreted Expressed in colon, small intestine, colonic tumors, bronchus, cervix and gall bladder . MUC3A Mucin 3A 7q22.1 Cell Surface Expressed in small intestine, colon, colonic tumors, heart, liver, thymus, prostate, Associated pancreas and gall bladder. MUC4 Mucin 4 3q29 Cell Surface Expressed in the thymus, thyroid, lung, trachea, esophagus, stomach, small intestine, Associated colon, testis, prostate, ovary, uterus, placenta, and mammary and salivary glands. MUC5AC Mucin 5AC 11p15.5 Oligomeric Secreted by gastric and respiratory tract epithelia, which protects the mucosa from Mucus/Gel‐ infection and chemical damage. Forming MUC6 Mucin 6 11p15.5 Oligomeric Expressed in the regenerative zone of gastric antrum, gastric body mucosa and Mucus/Gel‐ gastric incisura mucosa. Forming MUC7 Mucin 7 4q13.3 Oligomeric Expressed in mucous acinar cells of salivary gland tissues and thought to play a role Mucus/Gel‐ in removal of bacteria in the oral cavity aid in mastication, speech, and swallowing. Forming MUC8 Mucin 8 12q24.33 Cell Surface Expressed in the human airway mucin Associated MUC13 Mucin 13 3q21.2 Cell Surface Expressed in gastrointestinal and respiratory tracts Associated MUC15 Mucin 15 11p14.2 Cell Surface Spleen, thymus, prostate, testis, ovary, small intestine, colon, peripheral blood Associated leukocyte, bone marrow, lymph node and lung MUC17 Mucin 17 7q22.1 Oligomeric Expressed almost exclusively in the intestine. Mucus/Gel‐ Encodes a protein that functions as a cytoprotectant, maintains luminal structure, Forming and provide signal transduction MUC19 Mucin 19 12q12 Oligomeric Expressed by corneal epithelial cells, mucous cells of the submandibular gland and Mucus/Gel‐ submucosal gland and trachea middle ear epithelial cells. Forming MUC20 Mucin 20, Cell Surface 3q29 Expressed kidney, placenta, lung, prostate, liver, and digestive system. Associated

ICAM1 Intercellular Adhesion 19p13.2 Cell Surface Interacts with ntegrins of type CD11a / CD18, or CD11b / CD18 Molecule 1 (CD54) Associated ICAM2 Intercellular Adhesion 17q23.3 Cell Surface Interacts with ntegrins of type CD11a / CD18, or CD11b / CD18 Molecule 2 (CD102) Associated

ICAM3 Intercellular Adhesion 19p13.2 Cell Surface Interacts with ntegrins of type CD11a / CD18, or CD11b / CD18 Molecule 3 (CD50) Associated

ICAM4 Intercellular Adhesion 19p13.2 Cell Surface Landsteiner‐Wiener (LW) blood group antigen(s) and that shares similarity with the Molecule 4 Associated intercellular adhesion molecule (ICAM) protein family

ICAM5 Intercellular Adhesion 19p13.2 Cell Surface Expressed in Cerebral cortex, cerebellum, hippocampus, and lateral ventricle, and on Molecule 5, Telencephalin Associated the surface of telcephalic neurons and key for neuronal development

TSPAN4 Tetraspanin 4 11p15.5 Cell Surface Expressed in multiple tissues but is absent in brain, lymphoid cells, and platelets. Associated

TSPAN6 Tetraspanin 6 Xq22 Cell Surface Cytoplasmic and membranous staining was observed in most normal tissues. . Associated

TSPAN10 Tetraspanin 10 17q25.3 Cell Surface Expressed in the eye including iris, ciliary body, retinal pigment epithelium, but not Associated lens

TSPAN14 Tetraspanin 14 10q23.1 Cell Surface Expressed in the adrenal gland, Leydig cells, bone marrow, heart, spleen, neurons, Associated skeletal muscle

TSPAN15 Tetraspanin 15 10q22.1 Cell Surface Expressed in gall bladder, prostate and glandular cells Associated

TSPAN16 Tetraspanin 16 19p13.2 Cell Surface Expressed in pancreas, bronchus, gall bladder, renal tubules, fallopian tube, Leydig Associated cells, hematopoietic cells and myocytes.

TSPAN33 Tetraspanin 33 7q32.1 Cell Surface Expressed in pancreas, liver, breast, prostate, and neuronal cells Associated UPK1A Uroplakin 1A 19q13.12 Cell Surface Expressed peripheral nerves, umbrella cells in urothelium, thyroid gland, Purkinje Associated cells, stomach, and adrenal glandular cells. (TSPAN21)

ROM1 Tetraspanin 23 11q12.3 Cell Surface Expressed in the photo receptor disk rim of eye Associated

CD151 Tetraspanin 24 11p15.5 Cell Surface Expressed in blood vessels, and epidermis, but essential for proper assembly of the (Rod outer segment Associated glomerular and tubular basement membrane of the kidney membrane protein)

ELA cells were isolated from SF of OA paEents and the total RNA was extracted for microarrary analysis. (Chr. Chromosome) Jacobs Publishers 9 DNA microarray analysis

- overlap with other categories of stem cells, utilizing published gene datasets available from the NIH Gene Expression Omnibus. - pared with MSCs, we utilized high-density oligonucleotide To compare ELA cells and MSCs, we examined their respec- To verify that ELA cells are a distinct population of cells com- tive gene expression profiles in triplicate by microarray. The- correlation coefficient between these microarray datasets ob - microarrays and functional network analysis. DNA microar tained from repeated experiments was greater than 0.98, in and the results were compared with datasets from the NIH ray analysis was used to identify genes expressed in ELA cells, dicating high reproducibility. The entire set of expressed shows prothat tein-coding genes was used for a non-supervised hierarchical GEO and our laboratory (Figure 4B). This analysis revealed clustering analysis. The dendrogram in Figure 4A - the same tissue were found in different clusters, whereas the that 25% of the genes expressed by ELA cells were shared by freshly isolated and frozen/expanded ELA cells isolated from BM-derived MSCs (Figure 4B). The results were visually repre sented by a volcano plot to compare specific genes upregulated three categories of MSCs (BM-derived, CD105+, and CD133+) in ELA cells and MSCs (Figure. 4C). Additionally, we generated clustered together. These results suggested that ELA cells have Venn diagrams to compare gene expression in ELA cells and a gene expression profile distinct from MSCs. this analysis, we concluded that ELA cells are a distinct pop- ulationBM-derived, of ASCs. CD105+, and CD133+ MSCs (Figure 4D). From

DNA microarray analysis identified genes specific to MSCs, ESCs, and induced pluripotent stem cells (iPSCs) in the ELA cell population (Figure 4E). For example, ESCs expressed 1460 genes that were not expressed by the other stem cell types. ELA cells expressed unique genes as well as genes in common with ESCs, MSCs, and iPSCs (Table 4). Furthermore, ELA cells thesehad 616 genes genes play in commona role in withboth ESCs, cellular signifying function, a 42% such overlap as cell in the genetic profile of the cells. This finding is significant, as replication, and stem cell identity, such as self-renewal and cycle, RNA post-translation modification, cell death and DNA- ing pathways for DNA replication, recombination, and repair pluripotency. Additionally, IPA determined that shared signal - were significantly enriched between ELA cells, ESCs and iPSCs (right-tailed Fisher’s test, α=0.05), as shown in Table 4. Collec tively, our data suggests that ELA cells are functional ASCs with Figure 4. Comparison of gene expression profiles of early lin- categories of stem cells. a unique set of expressed genes that are not shared with other eage adult (ELA) cells and mesenchymal stem/progenitor cells (MSCs). Immunomodulatory capacity of ELA cells - Hierarchical cluster analysis of qRT-PCR data was performed on freshly isolated and cultured/expanded ELA cells and bone mar - row (BM)-derived, CD105+, and CD133+ MSCs. Expression levels There was no significant difference between irradiated and- were normalized to β-actin. Black represents 1, red represents >1, non-irradiated ELA cells co-cultured with fresh PBMCs in var- green represents <1, and grey represents below detection limits. ious ratios. Increasing the number of ELA cells did not influ ence the proliferative capacity of T cells in the co-culture (Fig (A) Dendrogram comparing gene expression in primary ELA cells,- BM-derived MSCs, CD105+ MSCs, and CD133+ MSCs. (B) Dendrogram ure 5A). In additional experiments, fresh and cryopreserved of a hierarchical cluster analysis comparing genes expressed in ex ELA cells were co-cultured with freshly isolated PBMCs from panded ELA cells against MSC gene data sets available from the NIH three healthy donors at a 1:10 ratio for 5 days (Figure 5B). No gene Expression Omnibus in addition to those generated in this study. significant proliferation of allo-reactive T cells was observed. - The SI was 1.72 (± 0.19; n=3) for fresh ELA cells and 1.29 (C) Volcano plots comparing specific genes upregulated in primary- ELA cells and expanded ELA cells (upper left), BM-derived MSCs (up (± 0.36; n=3) for cryopreserved cells. Treatment of ELA cells per right), CD105+ MSCs (bottom left), and CD133+ MSCs. Genes ex with the mitogen phytohemagglutinin showed no significant pressed above the broken red line represent genes specific to primary proliferation compared with the very active proliferation with- - freshly isolated PBMCs (Figure 5B). ELA cells at a ratio of 1:10 ELA cells on the left and genes specific to MSCs on the right. (D) Venn- diagram comparing upregulated genes in expanded ELA cells, BM-de elicited a moderate suppressive effect on CD3/CD28-stimu) rived MSCs, CD105+ MSCs, and CD133+ MSCs. (E) Venn diagram de lated, CFSE-labeled T cells (15.6 ± 2.7%; n=3) compared with picting the percentage of genes expressed in expanded ELA cells that a relatively low suppression effect by MSCs (2.8% ± 0.25; n=3 Jacobs Publishers 10

- (Figure 5C). ELA cells at passages 3, 5 and 6 suppressed CD3/- was 3.6 ± 0.36% when cultured with ELA cells (n=3) compared CD28-stimulated T lymphocytes, indicating that suppression with 1.6 ± 0.36% with PBMCs (n=3). Furthermore, a small pop- 5D).properties are maintained when ELA cells are expanded in cul ulation (< 1%) of T cells co-cultured with ELA cells expressed ture, although cells at passage 3 were most effective (Figure PD1 (Figure 5E-F). PD1 is generally associated with exhaus tion of T cells. We also found a modest increase in CD69, a Table 4. Common genes expressed in ELA, uESC, IPS, and MSCs together with enriched canonical pathways

ELA$vs.$uESC$Common$Genes$ ELA$vs.$IPS$Common$Genes$ ELA$vs.$MSC$Common$Genes$

Top$Molecular$and$Cellular$Func;ons$ Top$Molecular$and$Cellular$Func;ons! Top$Molecular$and$Cellular$Func;ons! $

! !Cell!cycle! ! !DNA!replica5on,!Recombina5on!and!Repair! ! !Cellular!Movement! ! !RNA!Post.Transcrip5onal!Modiﬁca5on! ! !Cell!cycle! ! !Cellular!Growth!and!Prolifera5on! ! !Cell!Death! ! !Cellular!Assembly!and!Organiza5on! ! !Cell!Death! ! !DNA!replica5on,!Recombina5on!and!Repair! ! !Cell!Death! ! !Cell!Morphology! ! !Cellular!Assembly!and!Organiza5on! ! !Cellular!growth!and!prolifera5on! ! !Cellular!Development!

Top$Physiological$System$Development$and$ Top$Physiological$System$Development$and$Func;on! Top$Physiological$System$Development$and$Func;on! Func;on$ $

! !Connec5ve!Tissue!Development!and!Func5on! ! !Connec5ve!Tissue!Development!and!Func5on! ! !Organismal!Development! ! !Cell.Mediated!Immune!Response! ! !Cell.Mediated!Immune!Response! ! !Tissue!Development! ! !Humoral!Immune!Response! ! !Humoral!Immune!Response! ! !Cardiovascular!System!Development!and!Func5on! ! !Cardiovascular!System!Development!and! ! !Organismal!survival! ! !!Connec5ve!Tissue!Development!and!Func5on! Func5on! ! !Tumor!Morphology! ! !!Skeletal!and!Muscular!System!Development!and! ! !Hepa5c!System!Development!and!Func5on! ! Func5on!

Top$Canonical$Pathways$$ Top$Canonical$Pathways$! Top$Canonical$Pathways$!

! !Cell!Cycle:!!G2/M!DNA!Damage!Checkpoint! ! !Parkinson!Signaling! ! !Hepa5c!Fibrosis/!Hepa5c!Stellate!Cell!Ac5va5on! Regula5on! ! !Tight!Junc5on!Signaling! ! !An5gen!Presenta5on!Pathway! ! !Pyrimidine!Metabolism! ! !Pyrimidine!Metabolism! ! !Oncosta5n!M!Signaling! ! !Wnt/B.Catenine!Signaling! ! !Role!of!BRCA1!in!DNA!Damage!Response! ! !IL.6!Signaling! ! !Tight!Junc5on!Signaling! ! !Cell!Cycle:!!G2/M!DNA!Damage!Checkpoint! ! !TREM1!Signaling! ! !Role!of!BRCA1!in!DNA!Damage!Response! Regula5on! !

Top$Networks$ Top$Networks$ Top$Networks!

! !Cell!Death,!Cancer,!Cellular!Movement! ! DNA!Replica5on,!Recombina5on,!and!Repair,!Cell! ! !Infec5on!Mechanism,!Cancer,!Gene!Expression! ! !DNA!Replica5on,!Recombina5on,!and!Repair,! Cycle,!Cancer! ! Cellular!Movement,!Cancer,!Cell.to.Cell!Signaling!and! Cell!Cycle,!Cancer! ! !DNA!Replica5on,!Recombina5on,!and!Repair,!Cell! Interac5on.! ! !Gene!Expression,!Cell!Cycle,!and!Cancer! Cycle,!Cancer! ! !Cellular!Growth!and!Prolifera5on,!Cancer,!Cell!Cycle! ! !Cell!Cycle,!Cancer,!Cellular!Growth!and! ! DNA!Replica5on,!Recombina5on,!and!Repair,!Cell! ! !Gene!Expression,!Cell!Death!,!Tissue!development! Prolifera5on! Cycle,!Cellular!Assembly!and!Organiza5on! ! !Gene!Expression,!Developmental!Disorder,!Gene5c! ! !! ! !Cellular!Assembly!and!Organiza5on,!RNA!Post.! Disorder! Transcrip5onal!Modiﬁca5on,!Cardiovascular!Disease! ! Gene!Expression,!Cell!Cycle,!Dermatological!disease! and!condi5on! ! Genes expressed in ELA cells were !! compared with publically available datasets from the NIH Gene Expression Omnibus: The Ingenuity Pathway Analysis (IPA) was used for idenEfying canonical pathways that were signiﬁcantly enriched (Right-‐tailed Fisher test with a = 0.05)

- - - Next, we wanted to determine if ELA cells promoted the expan surrogate marker of T cell responsiveness to mitogen and an tigen stimuli, in CD3+ T cells cultured with ELA cells (Figure- withsion ofappropriate CD4+/CD25+ antibodies, regulatory and T analyzedcells. ELA by cells bi-dimensional were co-cul tured with freshly isolated PBMCs for a period of 5 d, stained 5E-F). Taken together, these studies indicate that ELA cells might perform their immunosuppressive functions by inhibit FACS analysis. The percentage of CD4+ T cells expressing CD25 ing T cell proliferation and expanding CD4+/CD25+ regulatory - expanded 3-fold (10 ± 0.36%; n=3) when co-cultured with T cells. - ELA cells compared with PBMCs (1.6 ± 0.36%; n=3) (p=0.01) MSCs are known to inhibit the expression of activating recep (Figure 5E-F). The percentage of CD8+ T cells expressing CD25 tors on the surface of NK cells and potentially impair their cy Jacobs Publishers 11 - - totoxic activity. To evaluate potential ELA cell-mediated inhi ter 5 d culture, T cells were analyzed by flow cytometry to determine bition of NK cell lytic potential, we performed cytolytic assays. cellCFSE passage fluorescence. was determined T cell suppression by using wasdifferent expressed passage as thenumbers Prolifer of Purified populations of NK cells were co-cultured overnight- ation Index. (D) The immunosuppressive effectiveness of each ELA with and without ELA cells at a 1:1 ratio and than exposed to [51] Cr-labeled K562 target cells at various ratios. Cytolytic ac- cells co-cultured with CFSE-labeled T cells. (E) A bivariate dot plot tivity was measured by [51] Cr release. NK cells pre-incubated- analysis of a representative experiment of expanded ELA cell/freshly with ELA cells demonstrated >60% reduction in cytotoxic ef isolated PBMC co-culture (1:10 ratio) to determine the expression of fects. This reduction was consistent across decreasing concen CD25 on CD4+ and CD8+ T cells and the expression of CD69 or PD1 trations of NK cells (Figure 5G). These data suggested that ELA on CD3+ T cells (* p < 0.001; ** p < 0.01 as compared with MSCs). (F) cells have a suppressive effect on NK cells. Bar graphs represent the mean of 3 separate experiments ± SEM (* p < 0.001 as compared with PBMCs alone). (G) The inhibitory effect of ELA cells on NK cell cytotoxicity was demonstrated by co-culturing NK cells with or without ELA cells prior to incubation with target cells at various ratios. Discussion

Many groups have studied human BM stromal cells and demonstrated phenotypic and functional heterogeneity [22,31,32]. It cellhas nomenclaturebeen hypothesized used thatto refer naïve to stema heterogeneous cells have a populationgreater ca ofpacity marrow to differentiate stromal cells, into but functional it now justcells refers and tissue to MSCs, [33]. a Stem single type of ASCs. During early bone marrow studies, cells with

- self-renewing capacity were discovered and referred to as stem cells. These stem cells were later subdivided into hematopoietic and non-hematopoietic populations [34,35]. HSCs, one of earliest and best characterized BM stromal stem cell types, al lowed the first truly successful cellular therapies. To date, over 50,000 HSC transplants have been performed worldwide [36]. notNon-hematopoietic all marrow stromal MSCs cells were are commonly phenotypically identified or function by their- allyability identical, to differentiate and therefore into mesodermal not all are tissues capable [37]. of differenti However,-

of marrow stromal cells comes from studies of MSCs, leading ating into similar tissues [38-40]. Most of our understanding the bone marrow stroma that has a hierarchical relationship Figure 5. Immunomodulatory potential of early lineage adult many to believe that there is one category of ASC derived from- (ELA) cells. by co-culturing irradiated and non-irradiated ELA cells with fresh- with other ASCs [41]. However, within the bone marrow stro (A) Stimulation of allo-reactive T cells was determined ma there exists a heterogeneous population of ASCs, such as mesenchymal precursor cells (MPCs), marrow-isolated adult ly isolated peripheral blood mononuclear cells (PBMCs) at ratios of multilineage inducible (MIAMI) cells [42], multipotent adult 1:10, 1:100 and 1:1000 in triplicate for 5 d. PBMCs were isolated from aprogenitor population cells of ASCs(MAPCs) that [43], contribute and very to smallthe heterogeneity embryonic-like of healthy donors, and previously expanded ELA cells were irradiated. the(VSEL) marrow stem stromalcells [44]. cell Recently, population. our groupDespite isolated standardization ELA cells, To determine the proliferation of allo-reactive T cells, cultures were of protocols for stem cell biomanufacturing, many protocols pulsed with 3[H]-Thymidine (1 μCi/well) 18 h prior to harvesting. - start with a heterogeneous population that might not repre- Control background Thymidine uptake in PBMCs alone was (487 ± 62.9; n=5). Bar graphs represent the mean ± SEM of 5 separate exper address this concern, we plan to focus our future studies on iments. (B) The effectiveness of cryopreserved ELA cells to stimulate - sent the appropriate stem cell for a particular treatment. To- allo-reactive T cell proliferation was determined as in (A). Bar graphs represent the mean of 3 separate experiments ± SEM. (C) Immuno identifying subsets of the primitive stem cells based on mo suppressive properties were further investigated by culturing ELA - lecular profiles with the intention of clonally expanding them. cells or MSCs with 5-6-carboxyfluorescein diacetate succinidyl ester (CFSE)-labeled allo-reactive T cells at various ratios in triplicate. Af ELA cells represent a CD99+/MUC1+/CD235a-/MHC class I- Jacobs Publishers 12 - - erties of these cells were determined by protein and mRNA population from the SF of osteoarthritic patients. The prop numberes as a result and size of inflammationof bone canals, that which accompanies allow communication osteoarthri (embryonic form), REX1, and NANOG. Our isolation method tis. This inflammatory environment is known to increase the analysis for pluripotency genes and proteins, such as OCT4 in bone canal size may allow more ELA cells to migrate into the between the BM and the synovial cavity [46,48]. This increase was less cumbersome than those required for the enrichment are smaller in size and number, thus limiting the number of of stem cells from other tissues. The primary reason for this ELAjoint cells space. in theUnder joint non-inflammatory space. conditions, these canals CASYwas the Cell absence Counter of and RBCs Analyzer in the SF.system. Accurate ELA measurementsdiameter was 4-6 of ELA cell size, volume, and viability were performed with the- Our histochemical data suggests that ELA cells can differenti-

μm, in contrast to RBCs, which have a diameter of 6-8 μm. No tably, the majority of previous stem cell studies have utilized ate into various tissues. However, molecular profiling of these forward and side scatter perimeters on flow cytometry, which tissues showed that transcripts specific for other tissues are excludes cells < 6 μm. This gating strategy may explain why the phenotypealso expressed, that possessessome of them the abilityat high to levels. transdifferentiate This property into of ELA cell population has been overlooked until now. ASCs differentiated in vitro might represent an intermediate-

Our studies have shown that the genes ELA cells share with other tissue types. It is also possible that in vitro culture con MSCs are not the same as those shared with IPS cells or ESCs. ditions are not representative of in vivo conditions that induce ELA cells are similar in size to VSEL cells, which express OCT-4 terminal differentiation. These findings support a concept- and can differentiate into the three germ layers [44]. However, described by Quensenberry and colleagues suggesting that the ELA cell population does not express CXCR4, SSEA-4, CD34, expansion and differentiation of cells and changes in gene ex or CD133, which are additional markers used to identify the pression are continuous and reversible, and that sorting stem VSEL cell population [44]. Moreover, ELA cells proliferate in cells by static cell surface proteins may leave behind a large the absenceELA cell ofpopulation feeder cells, is distinct making from them the functionally VSEL cell populadistinct- percentageIt was determined of potential that stem MSCs cells possess [49]. immunomodulatory from VSEL cells. Taken together, these findings suggest that - tenance of peripheral tolerance, transplantation tolerance, au- tion. This raises the question of whether ELA cells represent a- properties [50,51] and might play specific roles in the main peuticheterogeneous strategies population for tissue ofrepair. primitive stem cells and whether We focused on the role of ELA cells in modulating the immune these subsets can be isolated and clonally expanded for thera toimmunity, tumor evasion, and fetal-maternal tolerance [50]. - do not induce an allo-immune response, suggesting that they - primarilyresponse by immunomodulate activating T cells. suppression We demonstrated by affecting that ELA the cells ef- In addition to the expression of the pluripotency gene tran- fector arm of the immune response. scripts NANOG, OCT4, REX-2, and DPPA3, ELA cells also ex press high levels of MUC1. Our transcriptome studies con ELA cellsNotably, did notour upregulatein vitro data a firmed high expression of mucin genes. Recent studies have suggests that ELA cells suppress T cell activation and induce shown a relationship between MUC1 and ESC differentiation regulatory T cells. Moreover, [45]. There is a need to distinguish the most primitive ASCs, surrogate marker of T-cell responsiveness (CD69) in CD3+ T- functionin particular as a ELAbetter cells, starting from populationmore mature for forms, stem cellas primitive therapy. cells [52]. ELA cells might interfere with T cell function in a- ASCs might possess broader differentiation capacity [33] and PD-1 independent pathway [45]. Although PD-1 was not up regulated in either CD4+ or CD8+ T cells, this does not pre- Future studies will involve the use of cell surface proteins such- dition,clude the ELA possibility cells were that shown ELA to cells inhibit secrete the cytolytic factors capacityor express of as MUC1 to distinguish between primitive and mature ASCs. cell surface proteins that may modulate T cell function. In ad cell-basedCollectively, treatment. our findings suggest that ELA cells might repre - sent a primitive form of ASCs, making them useful for stem- nateNK cells. immunity. Taken together, these findings suggest that ELA cells - evade the immune system by interfering with adaptive and in - - Although ELA cells reside in a dormant state in the SF (unpub - lished data), their origin is unclear. It is unlikely that SF con Our observations of the ELA cell population are of fundamen verts MSCs into ELA cells. We hypothesize that the origin of fortal importancestem cells that to the replace, field of regenerate, regenerative and medicine modulate and immune the de ELA cells is the BM and not the systemic circulation. We base velopment of cell therapeutics. There is an ever-growing need- this hypothesis on elegant studies performed by Nakagawa and - colleagues, who demonstrated that BM stromal cells migrate- function. However, relying on cell and tissue donation is unre directly from the BM to the joint space in a collagen-induced- liable and cannot address the need for ASCs. Biomanufactur- arthritis model [46,47]. We propose that the enhanced migra ing of ASC therapies is the most logical option [53]. Although tion of ELA cells into the joint space (synovial cavity) increas ASCs can be efficiently expanded in the laboratory, this expan Jacobs Publishers 13 sion cannot be easily translated to large-scale production for therapeutic purposes due to concerns, further studies are underway to assess whether pro- 4. 292(5819), 154-156 . technical issues. To address these- ic genes and ELA cell function. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, longed culture periods affect the expression of ELA cell-specif Swiergiel JJ et al. Embryonic stem cell lines derived from Conclusion 5. human blastocysts. Science. 1998, 282(5391):1145-1147.

Thomson JA, Kalishman J, Golos TG, Durning M,. Harris CP et al. Isolation of a primate embryonic stem cell line. Proc We have demonstrated the existence of a population of ASCs,- 6. Natl Acad Sci U S A. 1995, 92(17): 7844-7848 guishablereferred to from as ELAother stem ASCs. cells, ELA which cells are reside also withinmolecularly the SF dis of- osteoarthritic joints. These cells are phenotypically distin Keating, A. Mesenchymal stromal cells: new directions. 7. Cell Stem Cell. 2012, 10(6): 709-716. tinguishable from ESCs, IPS cells, and MSCs by their unique gene expression patterns. ELA cells represent a new form of Keating, A. Mesenchymal stromal cells. Curr Opin Hematol. 2006,Huang, 13(6): X., Cho, 419-425. S, Spangrude, G. J. Hematopoietic stem aprimitive promising ASCs tool with for in therapeutic vitro tissue approaches regenerative aimed capability at inhibi that- tionmight of benefit the immune regenerative response, therapies. ELA cells As withmight MSCs, also whichbe useful are 8. cells: generation and self-renewal. Cell Death Differ. 2007, patients undergoing allogeneic HSC transplantation or for the 14(11): 1851-1859. for preventing or suppressing graft-versus-host disease in treatment of certain autoimmune diseases. In conclusion, ELA receptors, hematopoiesis, and stem cells. Mol Endocrinol 9. Chute J p, Ross J R, McDonnell D P. Minireview: Nuclear cells have far reaching implications in both basic research and 10. 2010, 24(1): 1-10. regenerative medicine. Acknowledgements . Zon, L. I. Intrinsic and extrinsic control of haematopoietic 11. stem-cell self-renewal. Nature. 2008, 306-313 al. Long-term engraftment failure after marrow ablation The authors gratefully acknowledge the helpful advice provided Bentley SA, Brecher ME, Powell E, Serody JS, Wiley JM, et by Drs. Andrew Makarovskiy and John Garvey. In addition, we between peripheral blood stem cell and bone marrow re- gratefully acknowledge the help and assistance provided by the and autologous hematopoietic reconstitution: differences logistics staff for collecting and preparing synovial fluid samples for research purposes. Finally, we would like to acknowledge the 12. cipients. Bone Marrow Transplant. 1997, 19(6): 557-563. patients who participated in providing samples for the studies. al. Early detection of hematopoietic engraftment after Funding boneGreinix marrow HT, Linkesch and peripheral W, Keil F, blood Kalhs stem P, Schwarzinger cell transplanta I et- - Research reported in this publication was supported by the tion by highly fluorescent reticulocyte counts. Bone Mar 13. row Transplant . 1994, 14(2): 307-313. inDepartment study design, of Orthopedic data collection Surgery and atanalysis, Brigham decision and Women’s to pub- lish,Hospital or preparation and Asclepius of the Laboratories. manuscript. The funders had no role Sakaguchi, Y, Sekiya, I., Yagishita, K, Muneta, T. Comparison of human stem cells derived from various mesenchymal References tissues: superiority of synovium as a cell source. Arthritis 14. Rheum. 2005, 52(8): 2521-2529. 1. - et al. Multilineage potential of adult human mesenchymal Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R. Weissman, I. L. Translating stem and progenitor cell biolo gy to the clinic: barriers and opportunities. Science. 2000, 15. stem cells. Science. 1999, 284(5411): 143-147. 2. 287(5457):Martin, G. R. 1442-1446. Isolation of a pluripotent cell line from early Heterotopic of bone marrow. Analysis of precursor cells mouse embryos cultured in medium conditioned by ter- Friedenstein A.J, Petrakova, K.V, Kurolesova AI. Frolova GP.-

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