Cell Surface Marker Profile Research Articles

Mesenchymal stem cells derived from different origins have unique sensitivities to different chemotherapeutic agents

BMSCs (bone-marrow-derived mesenchymal stem cells) and ADSCs (adipose tissue-derived mesenchymal stem cells) are virtually identical in cell surface marker profile and differentiation potential. These cell populations have promising characteristics for clinical application. We have investigated the sensitivity of these cell populations to various chemotherapeutic agents by testing the inhibition of cell proliferation, low molecular DNA bands formation, in situ apoptosis, apoptosis-related gene expression and cell senescence after treatment. BU (busulfan), methotrexate and doxorubicin treatment led to a marked and dose-dependent reduction in cell viability compared with 5-FU (5-fluorouracil) treatment. Different expression patterns of apoptosis-related genes were found in the BMSCs and ADSCs following treatment with the agents, but no low molecular mass DNA bands were detected. BMSCs had a higher percentage of apoptotic and senescent cells following treatment with chemotherapeutic agents compared with ADSCs. These findings suggest that these two cell populations respond differently to chemotherapy treatment. ADSCs are more resistant than BMSCs to chemotherapy-induced senescence and apoptosis, indicating that they might be more advantageous to use in the clinic than BMSCs.

Comparison of bone marrow and adipose tissue-derived canine mesenchymal stem cells

BackgroundBone marrow-derived mesenchymal stem cells (BM-MSCs) and adipose tissue-derived mesenchymal stem cells (AT-MSCs) are potential cellular sources of therapeutic stem cells. MSCs are a multipotent population of cells capable of differentiating into a number of mesodermal lineages. Treatment using MSCs appears to be a helpful approach for structural restoration in regenerative medicine. Correct identification of these cells is necessary, but there is inadequate information on the MSC profile of cell surface markers and mRNA expression in dogs. In this study, we performed molecular characterization of canine BM-MSCs and AT-MSCs using immunological and mRNA expression analysis.ResultsSamples were confirmed to be multipotent based on their osteogenic and adipogenic differentiation. And these cells were checked as stem cell, hematopoietic and embryonic stem cell (ESC) markers by flow cytometry. BM- and AT-MSCs showed high expression of CD29 and CD44, moderate expression of CD90, and were negative for CD34, CD45, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. SSEA-1 was expressed at very low levels in AT-MSCs. Quantitative real-time PCR (qRT-PCR) revealed expression of Oct3/4, Sox2, and Nanog in BM- and AT-MSCs. There was no significant difference in expression of Oct3/4 and Sox2 between BM-MSCs and AT-MSCs. However, Nanog expression was 2.5-fold higher in AT-MSCs than in BM-MSCs. Using immunocytochemical analysis, Oct3/4 and Sox2 proteins were observed in BM- and AT-MSCs.ConclusionOur results provide fundamental information to enable for more reproducible and reliable quality control in the identification of canine BM-MSCs and AT-MSCs by protein and mRNA expression analysis.

Cryopreservation of umbilical cord mesenchymal cells in xenofree conditions

Mesenchymal stromal cells (MSC) are being used to treat and prevent a variety of clinical conditions. To be readily available, MSC must be cryopreserved until infusion. However, the optimal cryopreservation methods, cryoprotector solutions and MSC sensitivity to dimethyl sulfoxide (DMSO) exposure are unknown. This study investigated these issues. MSC samples were obtained from human umbilical cord (n = 15), expanded with Minimal Essential Medium-alpha (α-MEM) 10% human serum (HS), resuspended in 25 mL solution (HS, 10% DMSO, 20% hydroxyethyl starch) and cryopreserved using the BioArchive® system. After a mean of 18 ± 7 days, cell suspensions were thawed and diluted until a DMSO concentration of 2.5% was reached. Samples were tested for cell quantification and viability, immunophenotype and functional assays. Post-thaw cell recovery: 114 ± 2.90% (mean ± SEM). Recovery of viable cells: 93.46 ± 4.41%, 90.17 ± 4.55% and 81.03 ± 4.30% at 30 min, 120 min and 24 h post-thaw, respectively. Cell viability: 89.26 ± 1.56%, 72.71 ± 2.12%, 70.20 ± 2.39% and 63.02 ± 2.33% (P < 0.0001) pre-cryopreservation and 30 min, 120 min and 24 h post-thaw, respectively. All post-thaw samples had cells that adhered to culture bottles. Post-thaw cell expansion was 4.18 ± 0.17 ×, with a doubling time of 38 ± 1.69 h, and their capacity to inhibit peripheral blood mononuclear cells (PBMC) proliferation was similar to that observed before cryopreservation. Differentiation capacity, cell-surface marker profile and cytogenetics were not changed by the cryopreservation procedure. A method for cryopreservation of MSC in bags, in xenofree conditions, is described that facilitates their clinical use. The MSC functional and cytogenetic status and morphologic characteristics were not changed by cryopreservation. It was also demonstrated that MSC are relatively resistant to exposure to DMSO, but we recommend cell infusion as soon as possible.

Diabetes alters subsets of endothelial progenitor cells that reside in blood, bone marrow, and spleen

Circulating endothelial progenitor cells (EPCs) derived from the bone marrow (BM) participate in maintaining endothelial integrity and vascular homeostasis. Reduced EPC number and function result in vascular complications in diabetes. EPCs are a population of cells existing in various differentiation stages, and their cell surface marker profiles change during the process of mobilization and maturation. Hence, a generally accepted marker combination and a standardized protocol for the quantification of EPCs remain to be established. To determine the EPC subsets that are affected by diabetes, we comprehensively analyzed 32 surface marker combinations of mouse peripheral blood (PB), BM, and spleen cells by multicolor flow cytometry. Ten subsets equivalent to previously reported mouse EPCs significantly declined in number in the PB of streptozotocin-induced diabetic mice, and this reduction was reversed by insulin treatment. The PI(-)Lin(-)c-Kit(-)Sca-1(+)Flk-1(-)CD34(-)CD31(+) EPC cluster, which can differentiate into mature endothelial cells in vitro, was the highest population in the PB, BM, and spleen and occurred 61 times more in the spleen than in the PB. The cell number significantly decreased in the BM as well as in the PB but paradoxically increased in the spleen under diabetic conditions. Insulin treatment reversed the decrease of EPC subsets in the BM and PB and reversed their increase in spleen. A similar tendency was observed in some of the major cell populations in db/db mice. To the best of our knowledge, we are the first to report spatial population changes in mouse EPCs by diabetes in the blood and in the BM across the spleen. Diminished circulating EPC supply by diabetes may be ascribed to impaired EPC production in the BM and to decreased EPC mobilization from the spleen, which may contribute to vascular dysfunction in diabetic conditions.

Covering the Cover

Covering the Cover

Identification and characterization of chondrogenic progenitor cells in the fascia of postnatal skeletal muscle

Intramuscular injection of bone morphogenetic proteins (BMPs) has been shown to induce ectopic bone formation. A chondrogenic phase is typically observed in this process, which suggests that there may exist a chondrogenic subpopulation of cells residing in skeletal muscle. Two prospective cell populations were isolated from rat skeletal muscle: fascia-derived cells (FDCs), extracted from gluteus maximus muscle fascia (epimysium) and muscle-derived cells (MDCs) isolated from the muscle body. Both populations were investigated for their cell surface marker profiles (flowcytometry analysis), proliferation rates as well as their myogenic and chondrogenic potentials. The majority of FDCs expressed mesenchymal stromal cell markers but not endothelial cell markers. FDCs underwent chondrogenic differentiation after BMP4 treatment in vitro, but not myogenic differentiation. Although MDCs showed chondrogenic potential, they expressed the myogenic cell marker desmin and readily underwent myogenic differentiation in vitro; however, the chondrogenic potential of the MDCs is confounded by the presence of FDC-like cells residing in the muscle perimysium and endomysium. To clarify the role of the muscle-derived myogenic cells in chondrogenesis, mixed pellets with varying ratios of FDCs and L6 myoblasts were formed and studied for chondrogenic potential. Our results indicated that the chondrogenic potential of the mixed pellets decreased with the increased ratio of myogenic cells to FDCs supporting the role of FDCs in chondrogenesis. Taken together, our results suggest that non-myogenic cells residing in the fascia of skeletal muscle have a strong chondrogenic potential and may represent a novel donor cell source for cartilage regeneration and repair.

Fibrin Microbeads Loaded with Mesenchymal Cells Support Their Long-Term Survival While Sealed at Room Temperature

Efficient transfer of progenitor cells without affecting their survival is a key factor in any practical cell therapy. Fibrin microbeads (FMB) were developed as hard biodegradable cell carriers. The FMB could efficiently isolate mesenchymal stem cells (MSCs) from different sources and support the expansion of matrix-dependent cell types in a three-dimensional culture in slow rotation. The cells on FMB could also undergo induced differentiation for their eventual implantation to enhance tissue regeneration. FMB loaded with isolated human MSC (hMSC) were sealed in tubes topped up with medium. Almost full cell survival was recorded when the sealed cells were maintained in room temperature for up to 10 days, followed by a recovery period of 24 hrs at optimal conditions. Assay of cells recovery after such long room temperature storage showed ∼80%-100% survival of the cells on FMB, with only a marginal survival of cells that were kept in suspension without FMB in the same conditions. The hMSC that survived storage at room temperature preserved their profile of mesenchymal cell surface markers, their rate of proliferation, and their differentiation potential. The cell protective effect was not dependent on the presence of serum in the storage medium. It was clearly shown that over-expression of hypoxia induced factor-1α in hMSC with time, which may have protected the sealed cells on FMB at room temperature storage, was not necessarily related to extreme hypoxic stress. Foreskin normal fibroblasts on FMB sealed at room temperature were similarly protected, but with no elevation of their hypoxia-induced factor-1α expression. The results also show that FMB, unlike other commercially available cell carriers, could be used for delivery and shipping of progenitor cells at room temperature for extended time intervals. This could be highly useful for cell transfer for therapeutic application and for simplified cell transfer between different research centers.

Abstract 5202: Human colon cancer cell lines contain subsets of cells with the capacity to initiate highly prolific clonal growth in soft agar culture and to form transplantable tumor xenografts in vivo

Abstract Various studies underscore the key role of cancer ‘stem cells’ in the initiation of experimental tumor xenografts from cells derived from patient tumor specimens. Implantation of limited numbers of cancer cells – even ‘single cells’ – which exhibit putative cancer ‘stem cell’ markers were reported to form tumors in mice, although several months were required to achieve palpable tumor masses. In the current study we assessed whether established human colon cancer cell lines still possess the functional capacity to initiate tumor formation from ‘single cell’ preparations. Results indicate that each of seven colon cancer cell lines was capable of soft agar colony formation in RPMI-1640 medium containing either 5-10% FBS or 15% serum substitute. Inoculation of 96-well plates by single cell sorting resulted in highly prolific colony formation by a subset of cells derived from each cell line. While the large majority of colonies achieving a size &amp;gt;60μ in diameter exhibited growth arrest within 3 weeks (&amp;lt;&amp;lt;10 cell mass doublings), a minority of clones (&amp;lt;2%) exhibited sustained growth over the course of 3 – 8 weeks. Colony measurements and cell number / colony calculations indicated that these colonies accumulated cells exceeding 10 mass doublings. According to one ‘stem cell’ model of tumor growth (Mackillop et al, JNCI 70: 9-16, 1983), such prolific colony growth is attributed to the self-renewal of cancer ‘stem cells’, whereas limited colony growth is achieved by partially differentiated cells. To assess whether prolific colonies form tumors in vivo, 10 single colonies derived from each colon cancer cell line were implanted into each of 10 athymic nu/nu mice. Testing of 5 tumor models completed to date shows that 75 – 100% (86% overall) of these colonies form tumor xenografts (median times to 700 mg tumor formation ranged from 40 to 72 days). Subsequent in vivo passage of 3 tumor xenografts per cell line resulted in tumor formation in 85 – 92% of host animals (median times ranging from 29 to 67 days). Thus, despite extensive in vitro propagation, a subset of individual cells in each cell line retained the functional capacity to initiate and sustain cancer cell growth in vitro and in vivo. Single cell cloning in soft agar provides an effective means to isolate individual colonies formed by cancer ‘stem cells’ and to propagate human tumor xenografts with modest numbers of tumor cells, host mice, and tumor formation times. The cell surface marker profile and drug sensitivity of prolific colonies are currently under investigation. (Funded in part by NCI Contract No. HHSN261200800001E) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5202. doi:10.1158/1538-7445.AM2011-5202

Stem Cells: Their Role in Breast Cancer Development and Resistance to Treatment

About 20% of the total cells from primary breast tumors could generate palpable tumors in non-obese diabetic severe combined immunodeficient (NOD/SCID) immunocompromised mice. All the tumorigenic cells originate from a normal mammary stem cell. Human mammary stem cells are sensitive to oncogenic mutations and in mouse models they share similarities with breast cancer stem cells (BrCSCs). Tumorigenicity, invasion, progression and metastasization are further BrCSCs properties likely depending on their CD44+/CD24- phenotype. Local invasion and tumor metastasization seem to be facilitated by the epithelial to mesenchymal transition (EMT) program. This program may be reactivated from stable genetic alterations or through exposure of cancer cells to factors present in the surrounding micro-environment, or by an up-regulation of EMT-inducing transcription factors. One main explanation for resistance to treatment by cancer cells is that a rare subpopulation of cells in residual tumors with tumorigenic potential is intrinsically resistant to therapy. Consistent with this hypothesis, in human breast tumors, the subpopulation of tumor-initiating cancer cells with CD44(high)/CD24(low) cell surface-marker profile was found more resistant to cancer therapies (chemo, hormone and radiotherapy) than is the major population of more differentiated breast cancer cells. The reasons for CSC resistance to chemotherapy, hormone therapy and radiotherapy also have been examined and they opened new scenarios for cancer therapy.

TGF-β1 Accelerates Dendritic Cell Differentiation from Common Dendritic Cell Progenitors and Directs Subset Specification toward Conventional Dendritic Cells

Dendritic cells (DCs) in lymphoid tissue comprise conventional DCs (cDCs) and plasmacytoid DCs (pDCs) that develop from common DC progenitors (CDPs). CDPs are Flt3(+)c-kit(int)M-CSFR(+) and reside in bone marrow. In this study, we describe a two-step culture system that recapitulates DC development from c-kit(hi)Flt3(-/lo) multipotent progenitors (MPPs) into CDPs and further into cDC and pDC subsets. MPPs and CDPs are amplified in vitro with Flt3 ligand, stem cell factor, hyper-IL-6, and insulin-like growth factor-1. The four-factor mixture readily induces self-renewal of MPPs and their progression into CDPs and has no self-renewal activity on CDPs. The amplified CDPs respond to all known DC poietins and generate all lymphoid tissue DCs in vivo and in vitro. Additionally, in vitro CDPs recapitulate the cell surface marker and gene expression profile of in vivo CDPs and possess a DC-primed transcription profile. TGF-β1 impacts on CDPs and directs their differentiation toward cDCs. Genome-wide gene expression profiling of TGF-β1-induced genes identified instructive transcription factors for cDC subset specification, such as IFN regulatory factor-4 and RelB. TGF-β1 also induced the transcription factor inhibitor of differentiation/DNA binding 2 that suppresses pDC development. Thus, TGF-β1 directs CDP differentiation into cDCs by inducing both cDC instructive factors and pDC inhibitory factors.

2010 Nicolas Andry Award: Multipotent Adult Stem Cells from Adipose Tissue for Musculoskeletal Tissue Engineering

Cell-based therapies such as tissue engineering provide promising therapeutic possibilities to enhance the repair or regeneration of damaged or diseased tissues but are dependent on the availability and controlled manipulation of appropriate cell sources. The goal of this study was to test the hypothesis that adult subcutaneous fat contains stem cells with multilineage potential and to determine the influence of specific soluble mediators and biomaterial scaffolds on their differentiation into musculoskeletal phenotypes. We reviewed recent studies showing the stem-like characteristics and multipotency of adipose-derived stem cells (ASCs), and their potential application in cell-based therapies in orthopaedics. Under controlled conditions, ASCs show phenotypic characteristics of various cell types, including chondrocytes, osteoblasts, adipocytes, neuronal cells, or muscle cells. In particular, the chondrogenic differentiation of ASCs can be induced by low oxygen tension, growth factors such as bone morphogenetic protein-6 (BMP-6), or biomaterial scaffolds consisting of native tissue matrices derived from cartilage. Finally, focus is given to the development of a functional biomaterial scaffold that can provide ASC-based constructs with mechanical properties similar to native cartilage. Adipose tissue contains an abundant source of multipotent progenitor cells. These cells show cell surface marker profiles and differentiation characteristics that are similar to but distinct from other adult stem cells, such as bone marrow mesenchymal stem cells (MSCs). The availability of an easily accessible and reproducible cell source may greatly facilitate the development of new cell-based therapies for regenerative medicine applications in the musculoskeletal system.

Ultra scale‐down studies of the effect of shear on cell quality; Processing of a human cell line for cancer vaccine therapy

Whole cell therapy is showing potential in the clinic for the treatment of many chronic diseases. The translation of laboratory-scale methods for cell harvesting and formulation to commercial-scale manufacturing offers major bioprocessing challenges. This is especially the case when the cell properties determine the final product effectiveness. This study is focused on developing an ultra scale-down method for assessing the impact of the hydrodynamic environment on human cells that constitute the therapeutic product. Small volumes of a prostate cancer cell line, currently being developed in late phase II clinical trials as an allogeneic whole cell vaccine therapy for prostate cancer, were exposed to hydrodynamic shear rates similar to those present in downstream process, formulation and vial filling operations. A small scale rotating disc shear device (20 mL) was used over a range of disc speeds to expose cells to maximum shear rates ranging from 90 x 10(3) to 175 x 10(3) s(-1) (equivalent maximum power dissipation rates of 14 x 10(3) to 52 x 10(3) W kg(-1)). These cells were subsequently analyzed for critical cell quality attributes such as the retention of membrane integrity and cell surface marker profile and density. Three cell surface markers (CD9, CD147, and HLAA-C) were studied. The cell markers exhibited different levels of susceptibility to hydrodynamic shear but in all cases this was less than or equal to the loss of membrane integrity. It is evident that the marker, or combination or markers, which might provide the required immunogenic response, will be affected by hydrodynamic shear environment during bioprocessing, if the engineering environment is not controlled to within the limits tolerated by the cell components.

A tissue-like construct of human bone marrow MSCs composite scaffold supportin vivoectopic bone formation

Biocompatible and osteoconductive cell-scaffold constructs comprise the first and most important step towards successful in vivo bone repair. This study reports on a new cell-scaffold construct composed of gelatin-based hydrogel and ceramic (CaCO(3)/beta-TCP) particles loaded with human MSCs producing a tissue-like construct applied as a transplant for in vivo bone formation. Bone marrow-derived human MSCs were cultured in osteogenic induction medium. 5 x 10(5) (P(2)) cells were loaded on a mixture of hydrogel microspheres and ceramic particles, cultured in a rotating dynamic culture for up to 3 weeks. Both hydrogel microspheres and ceramic particles coalesced together to form a tissue-like construct, shown by histology to contain elongated spindle-like cells forming the new tissue between the individual particles. Cell proliferation and cell viability were confirmed by Alamar blue assay and by staining with CFDA, respectively. FACS analysis conducted before loading the cells, and after formation of the construct, revealed that the profile of cell surface markers remained unchanged throughout the dynamic culture. The osteogenic potential of the cells composing the tissue-like construct was further validated by subcutaneous transplants in athymic nude mice. After 8 weeks a substantial amount of new bone formation was observed in the cell-construct transplants, whereas no bone formation was observed in transplants containing no cells. This new cell construct provides a system for in vivo bone transplants. It can be tailored for a specific size and shape as needed for various transplant sites and for all aspects of regenerative medicine and biomaterial science.

Microarray-Based Characterization of a Colony Assay Used to Investigate Endothelial Progenitor Cells and Relevance to Endothelial Function in Humans

An assay proposed to quantify endothelial progenitor cell (EPC) colonies in humans was investigated to determine the phenotype of recovered cells and their relevance to in vivo endothelial function. Twelve sedentary subjects participating in a worksite wellness program underwent endothelial flow-mediated dilation (FMD) testing of the brachial artery and blood sampling for EPC colony assay. Microarray-based genotypic characterization of colonies showed surface markers consistent with T lymphocyte phenotype, but not with an EPC (CD34, CD133, VEGFR-2) or endothelial (CD146) phenotype. Gene expression patterns more closely matched T lymphocytes (r=0.87) than endothelial cells (r=0.66) in our microarray database. Flow cytometry of colonies confirmed large populations of CD3+CD45+ T cells (>75%) and few CD146+CD45- endothelial cells (<1%). Further, there was no correlation between colony number and the magnitude of FMD (r=-0.1512, P=0.6389). After exercise training, subjects improved FMD, from 6.7+/-2.0 to 8.7+/-1.9% (P=0.0043). Colonies also increased (P=0.0210), but without relation to FMD (r=0.1074, P=0.7396). T lymphocyte phenotype persisted after exercise (r=0.87). Cells in a commonly used EPC colony assay have a gene expression and cell surface marker profile consistent with a predominance of T lymphocytes and have an unclear relevance to endothelial function, either before or after exercise training.

Phenotypic Characterization and Invasive Properties of Synovial Fluid-Derived Adherent Cells in Rheumatoid Arthritis

The present study aimed at characterizing the phenotype and functions of adherent synovial fluid (SF) cells derived from rheumatoid arthritis (RA), comparing with fibroblast-like synoviocytes (FLS) derived from RA synovial tissue (ST). Adherent SF-derived cells were spindle-shaped from passages 1-6 under light microscopy. The cell surface marker profile in SF-derived cells from passage 1-6 was similar to that of ST-derived FLS. Levels of MMP-1 and MMP-3 were not significantly different between SF-derived cells and ST-derived FLS (p = 0.20 and p = 0.40, respectively). There was no significant difference in the optical density value between two cell types in the cell invasion assay (p = 0.10). SF-derived adherent cells have a fibroblast-like phenotype from very early culture passages and have the potential to produce MMPs with the invasive capacity to degrade cartilage, identical to ST-derived FLS. Therefore, these cells could substitute for ST-derived FLS in studying the pathogenesis of RA.

IL‐4 inhibits VLA‐4 expression on Tc1 cells resulting in poor tumor infiltration and reduced therapy benefit

We and others have previously demonstrated that IL-4-dependent Tc2 are inferior to Tc1-effector CD8+ T cells in regulating tumor progression in vivo. This functional disparity relates, in part, to the comparatively poor ability of Tc2 to migrate into diseased tissues. We now show that IL-4 treatment of committed Tc1 cells promotes the selective loss in the expression of very-late antigen (VLA)-4, without impacting the Tc1 cytokine production profile, cytotoxic activity, or expression of alternate cell surface markers. Down-regulation of VLA-4 expression on Tc1 cells was unique to treatment with IL-4 (i.e. Tc1IL-4) and did not occur in the presence of the Type-2 cytokine IL-13 or the regulatory cytokines IL-10 or TGF-beta. Notably, the inhibitory effects of IL-4 on Tc1 expression of VLA-4 could be blocked by the presence of IL-12, but not IFN-gamma. Predictably, Tc1IL-4 (but not Tc1 control) cells adhere poorly to plate-bound VCAM-1-Fc fusion protein and fail to be co-stimulated by VCAM-1 in vitro. They were also markedly impaired in their ability to traffic into intracranial melanoma lesions after adoptive transfer, yielding inferior therapeutic benefit to tumor-bearing mice. These results suggest a novel suppressive mechanism for IL-4 that limits Tc1 efficacy via preventing their recruitment into tumors.

Cytometric profiling in multiple sclerosis uncovers patient population structure and a reduction of CD8low cells

As part of a biomarker discovery effort in peripheral blood, we acquired an immunological profile of cell-surface markers from healthy control and untreated subjects with relapsing-remitting MS (RRMS). Fresh blood from each subject was screened ex vivo using a panel of 50 fluorescently labelled monoclonal antibodies distributed amongst 56 pools of four antibodies each. From these 56 pools, we derived an immunological profile consisting of 1018 'features' for each subject in our analysis using a systematic gating strategy. These profiles were interrogated in an analysis with a screening phase (23 patients) and an extension phase (15 patients) to identify cell populations in peripheral blood whose frequency is altered in untreated RRMS subjects. A population of CD8(low)CD4(-) cells was identified as being reduced in frequency in untreated RRMS subjects (P = 0.0002), and this observation was confirmed in an independent sample of subjects from the Comprehensive Longitudinal Investigation of MS at the Brigham & Women's Hospital (P = 0.002). This reduction in the frequency of CD8(low)CD4(-) cells is also observed in 38 untreated subjects with a clinically isolated demyelination syndrome (CIS) (P = 0.0006). We also show that these differences may be due to a reduction in the CD8(low)CD56(+)CD3(-)CD4(-) subset of CD8(low) cells, which have a natural killer cell profile. Similarities between untreated CIS and RRMS subjects extend to broader immunological profiles: consensus clustering of our data suggests that there are three distinct populations of untreated RRMS subjects and that these distinct phenotypic categories are already present in our sample of untreated CIS subjects. Thus, our large-scale immunophenotyping approach has yielded robust evidence for a reduction of CD8(low)CD4(-) cells in both CIS and RRMS in the absence of treatment as well as suggestive evidence for the existence of immunologically distinct subsets of subjects with a demyelinating disease.

Cells Isolated from the Epidermis by Hoechst Dye Exclusion, Small Size, and Negative Selection for Hematopoietic Markers Can Generate B Lymphocyte Precursors

Transdifferentiation has become a common claim for somatic stem cells, yet how such cells can be directed toward a specified cell lineage has not been well investigated. We previously demonstrated that when isolated epidermal stem cells were placed into an embryonic environment, their potential was extended beyond the keratinocyte lineage. Here, we present evidence that cells isolated using a modification of our published method for epidermal stem cells can be specifically directed to differentiate into B lymphocyte precursors. We found that these isolated cells co-cultured with S17 bone marrow stromal cells in cytokine-supplemented medium changed their cell surface marker profile and gene expression pattern to one characteristic of B lymphocyte precursors. Such cells also underwent variable, diversity, joining rearrangement at the immunoglobulin heavy-chain locus, a permanent genetic change unique to lymphocytes. This feature is limited to the cells isolated using the modified epidermal stem cell method, as cells isolated using the modified transit amplifying cell method could not be re-directed or reprogrammed. Such results demonstrate that cells from the epidermis can be directed to cross lineage boundaries to become mesodermally derived lymphocytes.

A comparative analysis of cytokine responses, cell surface marker expression and MAPKs in DCs matured with LPS compared with a panel of TLR ligands

Dendritic cells (DCs) are professional antigen-presenting cells that play a vital role in shaping adaptive immunity. DC maturation begins when exogenous danger signals bind to the appropriate toll-like receptor (TLR) and initiate expression of cell surface markers and the secretion of cytokines. This process occurs through defined mitogen-activated protein kinase (MAPK) signalling pathways. Of the 13 known mammalian TLRs, lipopolysaccharide (LPS), which activates TLR4, is the most commonly used ligand for the maturation of DCs in vitro. This comprehensive study measures cytokine secretion and cell surface marker expression in murine bone-marrow-derived DCs following maturation with LPS compared to DCs matured with a panel of other TLR-ligands (zymosan A (TLR2/6), PGN (TLR2), poly(I:C) (TLR3), flagellin (TLR5) and CpG-ODN1826 (TLR9)). The role of MAPK signalling pathways in the maturation process was also examined. Results demonstrate that zymosan A and CpG induce comparable cytokine and cell surface marker profiles to LPS. The remaining ligands differed significantly for cytokine and CD40 expression, but not for CD80 and CD86 expression. While there were differences for MAPK signalling pathways for all ligands, the effect of the inhibitors were broadly similar. These findings broaden our knowledge of TLR ligand-matured DCs.

Characterization of freshly isolated and cultured cells derived from the fatty and fluid portions of liposuction aspirates

Liposuction aspirates (primarily saline solution, blood, and adipose tissue fragments) separate into fatty and fluid portions. Cells isolated from the fatty portion are termed processed lipoaspirate (PLA) cells and contain adipose-derived adherent stromal cells (ASCs). Here we define cells isolated from the fluid portion of liposuction aspirates as liposuction aspirate fluid (LAF) cells. Stromal vascular fractions (SVF) were isolated separately from both portions and characterized under cultured and non-cultured conditions. A comparable number of LAF and PLA cells were freshly isolated, but fewer LAF cells were adherent. CD34+ CD45- cells from fresh LAF isolates were expanded by adherent culture, suggesting that LAF cells contain ASCs. Although freshly isolated PLA and LAF cells have distinct cell surface marker profiles, adherent PLA and LAF cells have quite similar characteristics with regard to growth kinetics, morphology, capacity for differentiation, and surface marker profiles. After plating, both PLA and LAF cells showed significant increased expression of CD29, CD44, CD49d, CD73, CD90, CD105, and CD151 and decreased expression of CD31 and CD45. Multicolor FACS analysis revealed that SVF are composed of heterogeneous cell populations including blood-derived cells (CD45+), ASCs (CD31- CD34+ CD45- CD90+ CD105- CD146-), endothelial (progenitor) cells (CD31+ CD34+ CD45- CD90+ CD105low CD146+), pericytes (CD31- CD34- CD45- CD90+ CD105- CD146+), and other cells. After plating, ASCs showed a dramatic increase in CD105 expression. Although some adherent ASCs lost CD34 expression with increasing culture time, our culture method maintained CD34 expression in ASCs for at least 10-20 weeks. These results suggest that liposuction-derived cells may be useful and valuable for cell-based therapies.