CD45low Cells Research Articles

In Acute Lymphoblastic Leukaemia, Stemness Is Frequent and Ubiquitous

AbstractAbstract 92Research on cancer stem cells, cells that self-renew and reconstitute the full phenotype of the original malignancy, has yielded controversial results regarding their frequency and identity for many cancers. The hierarchical stem cell model has been well established in some malignancies such as acute myeloid leukemia and states that only rare, immunophenotypically immature blasts harbor stem cell activity, resembling a normal physiological hierarchy. The opposing stochastic model proposes that stemness in cancer cells is supported by extrinsic stimuli and that a substantial fraction of malignant cells have this potential. Continued optimization of in vivo xenotransplantation modeling recently caused a paradigm shift for some cancers, for example in malignant melanoma where stem cell activity was found in as many as 1 in 4 cells. For acute lymphoblastic leukemia (ALL) we and others previously challenged the hierarchical model by demonstrating that both immature and more mature leukemic blasts contain self-renewal properties (Cancer Cell 2008, 14(1), p47-58). In this study we address the frequency of leukemic stem cells in the bulk leukemia and also, more specifically, in subpopulations of different blast maturity by using unsorted and highly purified flow sorted cell fractions. Primary patient material as well as leukemic blasts harvested from engrafted mouse bone marrow (secondary and tertiary material) were sorted for their CD10, CD20 or CD34 expression followed by orthotopic intrafemoral transplantation into severely immunocompromised NOD/scid IL2Rγnull (NSG) mice. Engraftment of transplanted CD19+CD10low and CD19+CD10high, CD19+CD20low and CD19+CD20high and CD19+CD34low and CD19+CD34high blast populations was monitored by 5 color flow cytometry using material from consecutive bone marrow punctures, final bone marrow harvests and/or single cell suspensions from spleens. Primary ALL samples from 15 high risk (BCR/ABL positive (n=8), BCR/ABL like ALL (n=2), high hyperdiploid/MRD positive (n=2), MRD positive (n=1), MLL/AF4 (n=2)), 3 intermediate risk (high WBC/MRD negative (n=2), age >10 years (n=1)) and 3 standard risk (n=3) patients were included. Cells sorted into CD19+CD10low and CD19+CD10high fractions were transplanted from primary patient material (n=4, HR; n=1, SR) and from secondary samples (n=4, HR; n=1; IR) with cells from one HR patient used at limiting dilutions. As few as 100 sorted cells of either fraction were sufficient to repopulate the leukemia. CD19+CD20high and CD19+CD20 low fractions from primary (n=7, HR; n=1, IR), secondary (n=5, HR; n=1, IR) and tertiary material (n=2, HR; n=1, IR) engrafted NSG mice. Limiting dilutions were performed on secondary (n=4, HR) and tertiary material (n=2, HR). Cell numbers required for engraftment varied between leukemias with as few as 100 cells being sufficient to cause engraftment. Limiting dilution experiments using CD19+CD34high and CD19+CD34low fractions from secondary (n=1, HR) and tertiary (n=1, HR) material yielded engraftment with as few as 10 CD19+CD34high and 100 CD19+CD34low cells. Similarly, unsorted primary (n=11, HR; n=2, IR), secondary (n=2, HR) and tertiary material (n=1, HR) required as few as 10 cells for leukemic reconstitution. Taken together, both unsorted and sorted blasts of all immunophenotypes and transplanted with low numbers were able to reconstitute the complete original phenotype of the patient leukemia. All limiting dilutions were transplanted down to 10 cells per mouse and those mice not engrafted yet are still under observation. Furthermore, the ability to self-renew was demonstrated by serial transplantation. Finally, we compared expression of self-renewal associated genes (BMI1, EZH2, HMGA2, MEIS1, TERT) in CD19+CD34low and CD19+CD34high fractions of 5 HR and 1 SR samples with that in cord blood. Interestingly, expression of these genes was not dependent on the CD34 status of the leukemic cells, whereas HMGA2, MEIS1 and TERT were upregulated in CD34+ cord blood cells. In summary we provide strong evidence for the stochastic cancer stem cell model in B precursor ALL by demonstrating that (i) a broad spectrum of blast immunophenotypes exhibit stem cell characteristics and (ii) that this stemness is highly frequent among ALL cells. Disclosures:No relevant conflicts of interest to declare.

CD49d Expression Identifies a Chronic Lymphocytic Leukemia (CLL) Subset with High Levels of Circulating CD34 +Cells Co-Expressing Endothelial Cell Markers.

Abstract 2329Poster Board II-306 Introduction:In chronic lymphocytic leukemia (CLL), CD49d, often in association with CD38, has been shown to mark a disease subset with poor prognosis. Functionally, both molecules act as counter-receptors for surface structures (i.e. VCAM-1/CD106 and CD31) usually expressed by the endothelial/stromal component of tumor micro-environment. We have recently identified a micro-environmental circuitry which involves CD38 triggering, and eventually determines an enrichment of the VCAM-1/CD106-expressing endothelial component detected in the context of CLL infiltrates found in bone marrow biopsies. Data was also provided that CD49d/VCAM-1 interactions are active in delivering pro-survival signals to CD49d-expressing CLL cells (Zucchetto et al, Cancer Res, 69, 4001, 2009). In this study, we investigated the amount of circulating progenitors with endothelial phenotype in CLL samples with different CD49d and CD38 expression levels. Methods:Peripheral blood (PB) samples from 91 CLL cases purposely selected with WBC>25,000/μl (B cells absolute lymphocyte count >10,000/μl) were evaluated by multiparametric flow cytometry for the absolute count of circulating CD34+ cells (ISHAGE protocol in single platform). Whenever possible (i.e. if a cluster of at least 100 CD34+ cells was detectable), a further characterization was performed (4-6 colours flow cytometry) for circulating endothelial cells (CEC), identified as a CD34+CD45low cell population co-expressing one of the following endothelial markers: CD309/VEGFR-2, CD144/VE-cadherin, CD106/VCAM-1 and CD146/Muc-18. CD49d and CD38 expression by CLL cells was considered positive if exceeding the standard cut-off value of 30% of positive cells. Results:PB absolute CD34+ cell counts were 7.5±7.5/μl in CD49d+ CLL (32 cases), vs. 3.3±2.7/μl in CD49d− CLL (59 cases; p=2.6×10−4), or 9.4±8.7/μl in CD49d+ CLL (30 cases) vs. 4.6±2.9/μl in CD49d− CLL (18 cases; p=0.004) when only cases phenotyped for CEC were considered. Furthermore, when samples were stratified also for CD38 expression, values of circulating CD34+ cells increased to 10.6±10.1/μl in CD38+CD49d+ CLL (11 cases) vs. 3.1±2.4/μl in CD38−CD49d− (51cases; p=1×10−5). Regarding the absolute quantification of CEC, a CD49d+ phenotype again marked the CLL subset with the highest CEC count, as identified by the expression of either the CD309/VEGFR-2 (CEC counts 1.7±2.3/μl in CD49d+ CLL vs. 0.5±0.5/μl CD49d− CLL; p=0.009) or the CD144/VE-cadherin (CEC counts 0.8±1/μl in CD49d+ CLL vs. 0.3±0.5/μl in CD49d− CLL; p=0.057) endothelial markers on CD34+CD45low cells. Notably, CEC from CD49d+ CLL expressed CD106/VCAM-1 in virtually all cells (1.6±2.4/μl), while the other marker of endothelial activation CD146/Muc-18 was detected in a fraction of CEC only (0.4±0.9/μl). Conclusions:CD49d and CD38 expression by CLL cells identify a disease subset with significantly higher number of both circulating CD34+ cells and CEC. This phenomenon could be explained considering several aspects: i) the sharing of common phenotypic markers between CLL cells and CD34+ progenitors, including CD38 and CD49d, which could be responsible for a displacement of CD34+ progenitors in the context of micro-environmental niches; ii) the known capacity of CLL cells, especially with a unmutated IGHV gene status and/or a CD38+CD49d+ phenotype to produce pro-angiogenic factors including Ang-2; iii) the rare PB cells expressing CD34 and CEC markers may represent CLL cell precursors with tumor-initiating cell features. Studies are currently ongoing to dissect among these hypotheses Disclosures:No relevant conflicts of interest to declare.

A New Role for the Human Placenta as a Hematopoietic Site Throughout Gestation

We investigated whether the human placenta contributes to embryonic and fetal hematopoietic development. Two cell populations--CD34(++)CD45(low) and CD34( +)CD45(low)--were found in chorionic villi. CD34(++) CD45(low) cells display many markers that are characteristic of multipotent primitive hematopoietic progenitors and hematopoietic stem cells. Clonogenic in vitro assays showed that CD34(++)CD45( low) cells contained colony-forming units-culture with myeloid and erythroid potential and differentiated into CD56(+) natural killer cells and CD19(+) B cells in culture. CD34(+)CD45(low) cells were mostly enriched in erythroid- and myeloid-committed progenitors. While the number of CD34(++)CD45(low) cells increased throughout gestation in parallel with placental mass. However, their density (cells per gram of tissue) reached its peak at 5 to 8 weeks, decreasing more than 7-fold from the ninth week onward. In addition to multipotent progenitors, the placenta contained intermediate progenitors, indicative of active hematopoiesis. Together, these data suggest that the human placenta is potentially an important hematopoietic organ, opening the possibility of banking placental hematopoietic stem cells along with cord blood for transplantation.

Varying Levels of CD34 Expression Correlate with Maintenance and Differentiation of HSC/MPP-Like Cell Line EML.

The HSC/MPP-like cell line EML has the capacity for multilineage (erythroid, myeloid, lymphoid) differentiation in vitro, and is functionally heterogeneous. Phenotypically, EML cell line consists of Sca-1+ c-kit+ Flk− CD34+ cells (80%) and Sca-1+ c-kit+ Flk− CD34− cells (20%), and resembles Lin− c-kit+ Sca-1+ Flk2− BM cells, which are also heterogeneous for CD34 expression. Functional analysis has revealed that purified CD34+ EML cells reproducibly contain the majority of progenitors capable of mutilineage differentiation in response to cytokines, whereas CD34− EML cells differentiated poorly or not at all. Since the CD34+ EML cell population can be divided into CD34low, CD34medium and CD34high subpopulations we investigated the correlation between the progenitor content and the level of CD34 expression. The CD34low, CD34med and CD34high EML cells were purified by FACS sorting, and their multilineage differentiation analyzed by CFC assays. Multiple CFC experiments have reproducibly shown that the progenitor frequency declines with a decrease in the level of CD34 expression on EML cells, with CD34high cells producing the highest and the CD34low cells producing the lowest number of colonies. These findings indicate that CD34+ EML cell population is functionally heterogeneous, and that the overall multilineage differentiation of EML cells strongly correlates with the CD34 expression levels. Since the majority of LTR-HSC reside within LKS Flk2− CD34− BM cells, and LKS Flk2− CD34+ BM cells are highly enriched for STR-HSC and MPPs, we tested whether CD34− EML cells are more primitive and give rise to CD34+ cells. The CD34− and CD34+ EML cells were purified (>99% pure) by sorting, cultured separately for 7 days, and cell aliquots from these cultures analyzed daily for CD34 expression. Four separate experiments have revealed that both CD34− and CD34+ EML cells can generate each other, suggesting that although the CD34− EML cells could be more primitive, a subpopulation of CD34+ cells retains the capacity to give rise to CD34− cells. Previous studies have shown that mouse LTR-HSC can alternate between CD34− “inactive” and CD34+ “activated” state, and that the heterogeneous population of LKS CD34+ BM cells contains activated LTR-HSC, STR-HSC and MPPs. Thus, we investigated how the level of CD34 expression correlates with the capacity of CD34+ EML cells to give rise to CD34− cells. Purified CD34low, CD34med and CD34high EML cells were cultured for 5 days, and the cells from each culture analyzed for CD34 expression. Remarkably, three separate experiments have revealed that CD34low EML cells have the highest capacity to give rise to CD34− EML cells, whereas CD34med and CD34high EML cells had very little capacity to generate CD34− EML cells. These findings suggest a model in which (a) CD34− EML cells are more primitive cells in an “inactive” (differentiate inhibited) state that generate CD34+ EML cells, and (b) the population of CD34+ EML cells is heterogeneous and consists of CD34med/high cells that can readily differentiate into multiple lineages, and CD34low cells that are in “activated” state and can revert back to the CD34− state. Cumulatively, these results support the notion that the maintenance and differentiation of EML cells is linked with the varying levels of CD34 expression. Therefore, EML cell line could represent a powerful in vitro model to study the linear differentiation of CD34− cells into CD34+ cells, and the mechanisms of reversible CD34 expression and activation of primitive hematopoietic cells.

RNA-Binding Protein Pum2 Promotes Self-Renewal and Suppresses Differentiation of Multipotent Hematopoietic Cells by Maintaining Them in Inactive CD34− State.

Over-expression of RNA-binding protein Pum2 supports maintenance and suppresses mutilineage differentiation of murine multipotent HSC/MPP-like cell line EML. The analysis of HSC and MPP markers has revealed that ∼80% of wt EML cells are Sca–1+c-kit+ Flk2− CD34+ and ∼20% are Sca–1+ c-kit+ Flk− CD34−. In contrast, the majority (80–95%) of EML cells over-expressing Pum2 (Pum2-EML) are Sca–1+ c-kit+ Flk− CD34−. Functional analysis has revealed that purified CD34+ wt EML and Pum2-EML cells reproducibly contained the majority of progenitors capable of mutilineage differentiation in response to cytokines, whereas CD34− wt EML and Pum2-EML cells differentiated poorly or not at all. The CD34+ wt EML cells can be divided into CD34low, CD34medium and CD34high subpopulations, and the analysis of their progenitor cell content has reproducibly shown that the majority of progenitors reside within CD34high and CD34med cells, whereas CD34low cells contain very small percentage of progenitors. These findings indicated that CD34+ wt EML cells are functionally heterogeneous, and that the overall multilineage differentiation of EML cells strongly correlates with the CD34 expression levels. Since the majority of LTR-HSC reside within LKS Flk2− CD34− BM cells, and LKS Flk2− CD34+ BM cells are highly enriched for STR-HSC and MPPs, we tested whether CD34− EML cells are more primitive and give rise to CD34+ cells. Multiple experiments have revealed that purified CD34− and CD34+ wt EML cells can generate each other in culture, suggesting that although the CD34− EML cells could be more primitive, a subpopulation of CD34+ cells retains the capacity to give rise to CD34− cells. Identical experiments have revealed that Pum2-EML cells are maintained predominantly as CD34− cells that generate a small population of CD34low cells. Further analysis has revealed that among CD34+ wt EML cells the CD34low cells have the highest capacity to give rise to CD34− EML cells. Previous studies have shown that mouse LTR-HSC can alternate between CD34− “inactive” and CD34+ “activated” state, and that the heterogeneous population of LKS CD34+ BM cells contains activated LTR-HSC, STR-HSC and MPPs. Thus, we have proposed a model in which the CD34− EML cells are more primitive cells in an “inactive” (differentiate inhibited) state that give rise to all CD34+ EML cells. On the other hand, the heterogeneous population of CD34+ EML cells consists of CD34med/high cells that can readily differentiate into multiple lineages, and CD34low cells that are in “activated” state and can revert back to the CD34− state. Cumulatively, these results support the notion that Pum2 protein supports proliferation and suppresses differentiation (two critical components of self-renewal) of multipotent hematopoietic cells by maintaining them in an inactive CD34− state. To start elucidating molecular differences between wt EML and Pum2-EML cells, and their CD34+ and CD34− subpopulations, we performed expression analysis using Agilent 44K microarrays. Out of 14,868 expressed genes, 760 exhibited significant differences in expression level between wt EML and Pum2-EML cells, and included Mmp14, Mpp1, Ripk3, Calmodulin 5, DDEF2 (Development and differentiation enhancing factor 2), C/EBPbeta, TXK tyrosine kinase, Egr3, Ermap, Eraf, TNFR1a, Jam4 and others. Among 760 differentially expressed genes, 146 genes exhibited >10-fold higher expression in wt EML cells, and 85 genes exhibited >10-fold higher expression in Pum2-EML cells.

Measurement of brain microglial proliferation rates in vivo in response to neuroinflammatory stimuli: Application to drug discovery

Microglial activation is emerging as an important etiologic factor and therapeutic target in neurodegenerative and neuroinflammatory diseases. Techniques have been lacking, however, for measuring the different components of microglial activation independently in vivo. We describe a method for measuring microglial proliferation rates in vivo using heavy water (2H2O) labeling, and its application in screening for drugs that suppress neuro-inflammation. Brain microglia were isolated by flow cytometry as F4/80+, CD11b+, CD45(low) cells, and 2H enrichment in DNA was analyzed by gas chromatography/mass spectrometry. Basal proliferation rate was approximately 1%/week and systemic administration of bacterial lipopolysaccharide (LPS) markedly increased this rate in a dose-dependent manner. Induction of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice by MOG(35-55) peptide stimulated proliferation of CD45(low) microglia, which could be distinguished from the proliferation of CD45(high) infiltrating monocytes. Minocycline (45 mg/kg/day, i.p.) inhibited resident microglial proliferation in both the LPS and EAE models. Thirteen drugs were then screened for their ability to inhibit LPS-stimulated microglia proliferation. Female C57BL/6 mice were given LPS (1 mg/kg), and concomitant drug treatment while receiving 2H2O label for 7 days. Among the drugs screened, treatment with isotretinoin dose-dependently reduced LPS-induced microglial proliferation, representing an action of retinoids unknown previously. Follow-up studies in the EAE model confirmed that isotretinoin not only inhibited proliferation of microglia but also delayed the onset of clinical symptoms. In conclusion, 2H2O labeling represents a relatively high-throughput, quantitative, and highly reproducible technique for measuring microglial proliferation, and is useful for screening and discovering novel anti-neuroinflammatory drugs.

Measurement Of Brain Microglial Proliferation Rates In Vivo: A Biomarker For Discovering Novel Anti‐Neuroinflammatory Agents

The objective of this study was to screen drugs in vivo for inhibition of neuroinflammation, using a recently developed stable isotope method. Microglia (MG), the resident immune cells of the brain, mediates neuroinflammation and contributes to the pathogenesis of many diseases, including Alzheimer’s, multiple sclerosis and stroke. MG proliferation was measured by incorporation of 2H from heavy water (2H2O) into the deoxyribose moiety of DNA, after administration of lipopolysaccharide (LPS, 1 mg/kg, i.p). Follow-up studies were in the experimental autoimmune encephalomyelitis (EAE) model. MG were isolated from the brain, as CD11b+ cells (LPS model) or as F4/80+/CD45low cells by flow cytometry (EAE model). Male mice were treated with drugs of different classes for 7 days while receiving 2H2O (8% in drinking water). Minocycline (45 mg/kg, i.p.) and dexamethasone (5 mg/kg) inhibited MG proliferation. In the initial screen of drugs, treatment with 13-cis-retinoic acid (Accutane®, 40 mg/kg) significantly decreased LPS-induced MG proliferation. Follow-up studies in EAE confirmed inhibition of MG proliferation. Etiocholanedione combined with low-dose minocycline (30 mg/kg) inhibited MG proliferation. No effects were seen with aspirin, calcitriol, clofibrate, enalapril, ibuprofen, imipramine, ketoconazole or rosiglitazone. In summary, 2H2O labeling of DNA is a high-throughput, quantitative and highly reproducible method for measuring MG proliferation, and is useful for screening and discovering novel anti-neuroinflammatory drugs. Support: KineMed, Inc.

Expression of CD24 on CD19 −CD79a + early B-cell progenitors in human bone marrow

CD24 is a surface marker expressed in immature and mature B cells and involved in cellular adhesion and apoptosis. There are no data, which delineate the stage in early development of human B cells, which marks the expression of CD24. We studied lymphopoiesis in normal pediatric bone marrow (BM) and found that 1.5 ± 0.2% of WBC were CD24 + lymphocytes which did not express CD19. A significant fraction of these cells expressed low levels of CD45 (CD19 −CD24 +CD45 low cells). Small numbers of CD19 −CD24 +CD45 low cells were found in the regenerating BM of children with acute lymphoblastic leukemia after the completion of chemotherapy and in normal adult BM. Flow cytometric analyses have shown that CD19 −CD24 +CD45 low lymphocytes express CD10, CD34, CD79a, CD179a (VpreB), and TdT markers, i.e., displayed antigenic properties of early B-cell progenitors. Our data indicate that CD19 − early B-cell progenitors in human BM express CD24, and that the expression of CD24 in human B-cell development precedes the expression of CD19.

Flow cytometric analyses on lineage-specific cell surface antigens of rat bone marrow to seek potential myelotoxic biomarkers: status after repeated dose of 5-fluorouracil.

Flow cytometry (FCM) analysis of CD45, CD45R, CD71 and CD90 expression on Crj:CD(SD)IGS rat bone marrow cells was done after 5-fluorouracil (5-FU) administration to examine whether these lineage-specific cell surface antigens could be myelotoxic biomarkers. The expression of CD45 (CD45Low and CD45High: differing in expression intensity), CD45R, CD71 and CD90 on bone marrow cells coincided with previous reports. After repeated administration of 5-FU at 50 mg/kg/day for 1-5 days, a time-dependent decrease in cells expressing CD45Low, CD71 and CD90 was observed, whereas a decrease in the CD45High expressing cells was not observed. Furthermore, the decrease was dose-dependent in CD45Low, CD71 and CD90 expressing cells after administration of 5-FU between 2 and 50 mg/kg/day for 4 days. After 4-day repeated dose of 5-FU at 50 mg/kg/day followed by a recovery period, the change in number of CD45Low, CD45R, CD71 and CD90 cells to the bottom and in recovery showed different kinetics. In contrast, the change in number of CD45High cells was minimal, and relatively stable after 5-FU administration. The results suggest that CD45, CD45R and CD90 could each be potential myelotoxic biomarkers for a total proportion of common leukocytes including T- and B-lymphocytes, for a total proportion of B-lymphocytes, and for a total proportion of T-lymphocytes plus immature B-lymphocytes and common progenitor cells, respectively. CD71 could be a single myelotoxic biomarker for erythroid cells. Further study is required for isolation of each of the myelo-lymphocytic lineages. However, the present study showed that FCM analysis could be available to assess the lineage or differentiation stage-specific response, such as the different extent and time-course or the kinetics (the time to reach the bottom and to recover to the normal level) of myelotoxic effect in rat bone marrow.

A cellular model for myeloma cell growth and maturation based on an intraclonal CD45 hierarchy.

Multiple myeloma (MM) is a plasma cell malignancy mainly characterized by the accumulation of malignant plasma cells within the bone marrow. This review shows that the biology of CD45 illuminates that of MM and, more specifically, provides a better delineation of a tumor cell 'hierarchy' of clinical interest. We show that in MM, as in normal plasma cell differentiation, there is an intraclonal CD45 hierarchy that is a gradient of CD45 expression on myeloma cells directly related to their proliferation rate and differentiation status. This CD45 hierarchy allows for the design of a cellular model for MM-cell growth and maturation in which CD45 bright myeloma cells represent the proliferating compartment and CD45 low myeloma cells the quiescent compartment. This model includes an aberrant phenotype that is annihilation rather than decline of CD45, annihilation reflecting the terminal phase of the disease and/or an aggressive presentation of MM. Data from the literature suggest that CD45 bright myeloma cells are targeted by interleukin (IL)-6, whereas CD45 negative myeloma cells with a high clonogenic capacity are targeted by insulin/insulin-like growth factor 1 (IGF-1). This model will be useful for both a better understanding of the basic biology of MM and a better stratification of and therapeutic approach to the patients. Finally, this model presents MM as a self-renewing plasma cell disease, although the first oncogenic events such as 14q32 translocations clearly occur earlier in a B cell.

Isolation and characterization of bone marrow multipotential mesenchymal progenitor cells.

There is an increased interest in rheumatology in mesenchymal progenitor/stem cells (MPCs) and their roles in rheumatic diseases, but little is known about the phenotype of these cells in vivo. The aim of this study was to isolate and characterize human bone marrow (BM) MPCs. Fluorescence microscopy was used to identify putative MPCs among adherent BM cells. To purify them, a positive selection with antifibroblast microbeads was used, combined with fluorescence-activated cell sorting (FACS) for microbead+,CD45(low) cells. A more detailed phenotype of these cells was determined using 4-color flow cytometry, and standard chondrogenic, osteogenic, and adipogenic assays were used to investigate their differentiation potentials. Putative MPCs microscopically identified as large, fibroblast-like, D7-FIB+ cells were purified using positive selection with D7-FIB-conjugated (antifibroblast) microbeads followed by FACS for specifically bound microbead+,CD45(low) cells. These cells represented 0.01% of mononuclear cells in the BM. They were uniformly positive for CD105, LNGFR, HLA-DR, CD10, CD13, CD90, STRO-1, and bone morphogenetic protein receptor type IA (BMPRIA) and were negative for CD14, CD34, CD117, and CD133. Only cells with this phenotype could proliferate and produce adherent cell monolayers capable of chondrogenic, osteogenic, and adipogenic differentiation. D7-FIB- cells in the BM lacked any MPC activity. Uncultured skin fibroblasts had a phenotype similar to that of BM MPCs, but were negative for LNGFR, STRO-1, HLA-DR, and BMPRIA. This study shows the distinct phenotype, morphology, and method of isolation of BM MPCs. The findings may have implications for defining the physiologic roles of MPCs in arthritis, bone diseases, and joint regeneration.

Down-regulation of cell-surface CD4 co-receptor expression and modulation of experimental allergic encephalomyelitis.

Pre-immunization with autoantigens confers resistance in experimental models of autoimmune diseases. Since non-self molecules can also be protective, it is conceivable that part of the effect rests on a non-specific attenuation of the immune response. This study is aimed at identifying mechanisms by which pre-immunization with a moiety suspended in incomplete Freund's adjuvant (IFA) protects from experimental allergic encephalomyelitis (EAE). Lewis rats were immunized with each of either concanavalin A, lipopolysaccharide, bovine serum albumin, 70 or 65 kDa heat shock proteins, or myelin basic protein. All moieties were given in IFA 3 weeks prior to EAE induction. Serial cytofluorimetric monitoring of B cells and of the alpha beta TCR+, CD4+, CD8+, CD45high and CD45low cells was performed. IFN-gamma and IgG1 production was evaluated in parallel. All moieties were able to attenuate or abrogate the clinical signs of EAE. At day 4 and 10 after EAE induction, the surface expression of the CD4 molecule was down-regulated on T lymphocytes. This down-regulation was most evident in animals with the highest degree of clinical protection. By day 21 post-immunization, CD4 expression was restored. The same animals also showed an increase in the B cell percentage and Th2-related IgG1 production while IFN-gamma secretion was reduced. Pre-immunization with diverse antigens suspended in IFA confers resistance to EAE induction. The down-regulation of the CD4 co-receptor accompanied by events suggestive of an immune deviation may be a general mechanism that contributes to the protection.

Isolation and direct characterization of resident microglial cells from the normal and inflamed central nervous system.

In addition to the major population of infiltrating leukocytes recovered from inflamed rat central nervous system (CNS), all of which expressed high levels of leukocyte common antigen CD45, many cells were coisolated that were MRC OX42+ (complement receptor 3/CD11b) but expressed low-to-moderate levels of CD45 and major histocompatibility complex (MHC) class I molecules. Most cells from normal CNS, in contrast, lay within this latter, CD45low population. From previous in situ immunohistochemical studies, the fortuitously isolated CD45low cells were probably resident (ramified) microglia. Using irradiation chimeras, we show that resident microglia respond to inflammation by upregulating CD45, CD4, and MHC class I molecules with a minority of these cells increasing their expression of MHC class II molecules. A 3- to 4-fold increase in the number of microglia isolated from inflamed CNS provided indirect evidence that the cells had proliferated. In normal CNS, a very small population of blood-derived CD45high-expressing cells are present; most MHC class II expression is associated with these few cells and not with the resident microglia.