Interactions between monocytes, mesenchymal stem cells, and implants evaluated using flow cytometry and gene expression.

Monocytes and mesenchymal stem cells (MSC) are evident at the implants during early healing. However, when coexisting, their interactions at different implants have not been determined. This study uses an in vitro system, consisting of monoculture and direct co-cultures of monocytes and MSC on screw-shaped machined and oxidized titanium implants in combination with scanning electron microscopy, enzyme-linked immunosorbent assay, flow cytometry, cell sorting, and quantitative polymerase chain reaction. The cell-specific adhesion and gene expression of monocytes and MSC was determined. After 24 hr, the coexistence of monocytes and MSC in co-culture led to equal proportions of adherent monocytes and MSC, irrespective of the implant type. In contrast, higher number of adherent monocytes than MSC was found on the oxidized implant in monoculture. Quantitative polymerase chain reaction analysis of fluorescent activated cell sorting-sorted cells revealed up-regulation of interleukin-1beta, in monocytes, and interleukin-1beta and C-X-C chemokine receptor type 4, in MSC, when the cell types coexisted compared with monocultures. Further, in co-culture, the expression of bone morphogenetic protein-2, stromal cell-derived factor 1, and integrin-ß1 was enhanced in the implant-adherent MSC, but not monocytes. It is concluded that during the first 24 hr in an in vitro static condition, the effect of co-culture of monocytes and MSC was more prominent than the effect of the implant surface properties. The results indicate that the coexistence of monocytes and MSC on an implant alters the adhesion and expression of some genes compared with when each cell type existed alone. Further, the results show that the gene expression of major growth and recruitment factors is mainly enhanced in the implant-adherent MSC in contrast to implant-adherent monocytes in co-culture.

Cell viability and chondrogenic differentiation capability of human mesenchymal stem cells after iron labeling with iron sucrose.

For evaluation of cell therapy strategies using human mesenchymal stem cells (hMSCs), it is important to be able to trace transplanted cells and their distribution in tissues, for example, cartilage, over time. The aim of the study was to determine effects on cell viability, traceability, and chondrogenic differentiation of hMSCs after iron labeling with iron sucrose. hMSCs were collected (seven donors, 13-57 years) from patients undergoing spinal surgery. Two subsets of experiments were performed. (1) Iron labeling of hMSCs: 1 mg/mL of Venofer(®) (iron sucrose) was added (16 h) to cultures. hMSCs were examined for uptake of iron sucrose (Prussian blue staining) and cell viability (flow cytometry). (2) Iron-labeled hMSCs (passage 4) (n=4, pellet mass), 200,000 cells/tube, were cultured (DMEM-HG) with 10 ng/mL TGFß and compared with controls (from each donor). The pellets were harvested at days 7, 14, and 28. Real-time PCR, IHC, and histology were used to evaluate SOX9, ACAN, C6S, and COL2A1 expression. Mean number of cells containing iron deposits was 98.1% and mean cell viability was 92.7% (no significant difference compared with unlabeled control cells). Pellets containing iron-labeled cells expressed COL2A1 on protein level (all time points), in similar levels as controls, and glycosaminoglycan accumulation was observed in iron-labeled pellets (day 14 or day 28). Results were supported by the expression of chondrogenic genes SOX9, ACAN, and COL2A1. The results in vitro indicate that iron sucrose can be used as a cell tracer for evaluation of cellular distribution in vivo after transplantation of MSCs and thus contribute with important knowledge when exploring new treatment strategies for degenerated cartilaginous tissues.

Similar cellular migration patterns from niches in intervertebral disc and in knee-joint regions detected by in situ labeling: an experimental study in the New Zealand white rabbit.

INTRODUCTION: Potential stem cell niches (SNs) were recently reported in intervertebral discs (IVDs) and knee joints (KJs) in different mammals (located adjacent to the epiphyseal plate; EP). The aim here was to examine further possible cellular migration and migration directions of cells originating from niches possibly involved in regeneration of cartilaginous tissues in the IVD and in the KJ regions in adult mammals. METHODS: In total, 33 rabbits were used in studies A through C. A. IVD cells were sorted; fluorescence-activated cell sorting (FACS) by size (forward scatter; ≤ 10 µm or >10 µm or GDF5+ cells (anti-GDF5 antibody). Sorted cells, labeled with cell tracer (carboxyfluorescein-diacetate-succinimidyl ester; CDFA-SE) were applied on IVD explants in vitro. Migrating cells/distance was evaluated by fluorescence- and confocal-microscopy (FC). B. DNA labeling was performed with BrdU (oral administration). Animals were killed (14 to 56 days), KJs collected, and BrdU+ cells visualized with immunohistochemistry (IHC)/anti-BrdU antibody in SN and articular cartilage (AC). C. Cell tracer: (Fe-nanoparticles: Endorem) were injected into SNs of IVDs (LI-LV) and KJs (tibia). Animals were killed after 2 to 6 weeks. Fe-labeled cells were traced by ferric-iron staining (Prussian blue reaction; Mallory method). RESULTS: A. GDF5+ cells and ≤ 10-µm cells displayed the best migration capability in IVD explants. GDF5+ cells were detected at a tissue depth of 1,300 µm (16 days). B. BrdU+ cells were observed in early time points in niches of KJs, and at later time points in AC, indicating a gradual migration of cells. C. Fe+ cells were detected in IVDs; in annulus fibrosus (AF) in 11 of 12 animals and in nucleus pulposus (NP) in two of 12 animals. In AC (tibia), Fe+ cells were detected in six of 12 animals. In the potential migration route (PMR), from niches toward the IVD, Fe+ cells (three of 12 animals) and in PMR toward AC (KJs) (six of 12 animals) were detected. CONCLUSIONS: Results indicate similar cellular migration patterns in cartilage regions (IVD and KJs) with migration from stem cell niche areas into the mature cartilaginous tissues of both the KJs and the IVD. These findings of a cellular migration pattern in mature cartilage are of interest from tissue-repair and engineering perspectives.

Distinct transcriptional control in major immunogenetic subsets of chronic lymphocytic leukemia exhibiting subset-biased global DNA methylation profiles.

Chronic lymphocytic leukemia (CLL) can be divided into prognostic subgroups based on the IGHV gene mutational status, and is further characterized by multiple subsets of cases with quasi-identical or stereotyped B cell receptors that also share clinical and biological features. We recently reported differential DNA methylation profiles in IGHV-mutated and IGHV-unmutated CLL subgroups. For the first time, we here explore the global methylation profiles of stereotyped subsets with different prognosis, by applying high-resolution methylation arrays on CLL samples from three major stereotyped subsets: the poor-prognostic subsets #1 (n = 15) and #2 (n = 9) and the favorable-prognostic subset #4 (n = 15). Overall, the three subsets exhibited significantly different methylation profiles, which only partially overlapped with those observed in our previous study according to IGHV gene mutational status. Specifically, gene ontology analysis of the differentially methylated genes revealed a clear enrichment of genes involved in immune response, such as B cell activation (e.g., CD80, CD86 and IL10), with higher methylation levels in subset #1 than subsets #2 and #4. Accordingly, higher expression of the co-stimulatory molecules CD80 and CD86 was demonstrated in subset #4 vs. subset #1, pointing to a key role for these molecules in the crosstalk of CLL subset #4 cells with the microenvironment. In summary, investigation of three prototypic, stereotyped CLL subsets revealed distinct DNA methylation profiles for each subset, which suggests subset-biased patterns of transcriptional control and highlights a key role for epigenetics during leukemogenesis.

Tumour-loaded α-type 1-polarized dendritic cells from patients with chronic lymphocytic leukaemia produce a superior NK-, NKT- and CD8+ T cell-attracting chemokine profile.

Tumour-loaded dendritic cells (DCs) from patients with chronic lymphocytic leukaemia (CLL) matured using an α-type 1-polarized DC cocktail (IL-1ß/TNF-α/IFN-α/IFN-γ/poly-I:C;αDC1) were recently shown to induce more functional CD8(+) T cells against autologous tumour cells in vitro than DCs matured with the 'standard' cocktail (IL-1ß/TNF-α/IL-6/PGE(2) ;PGE(2) DCs). However, the ability of vaccine DCs to induce a type 1-polarized immune response in vivo probably relies on additional features, including their ability to induce a CXCR3-dependent recruitment of NK cells into vaccine-draining lymph nodes. Moreover, their guiding of rare tumour-specific CD8(+) T cells to sites of DC-CD4(+) T cell interactions by secretion of CCL3 and CCL4 is needed. We therefore analysed the chemokine profile and the lymphocyte-attracting ability in vitro of monocyte-derived PGE(2) DCs and αDC1s from patients with CLL. αDC1s produced much higher levels of CXCR3 ligands (CXCL9/CXCL10/CXCL11) than PGE(2) DCs. Functional studies further demonstrated that αDC1s were superior recruiters of both NK and NKT cells. Moreover, αDC1s produced higher levels of CCL3/CCL4 upon CD40 ligation. These findings suggest that functional αDC1s, derived from patients with CLL, produce a desirable NK-, NKT- and CD8(+) T cell-attracting chemokine profile which may favour a guided and Th1-deviated priming of CD8(+) T cells, supporting the idea that αDC1-based vaccines have a higher immunotherapeutic potential than PGE(2) DCs.

Effects of intervertebral disc cells on neurite outgrowth from dorsal root ganglion explants in culture.

STUDY DESIGN: An experimental study investigating the effect of disc cells on neurite outgrowth in a rat dorsal root ganglion (DRG) culture system. OBJECTIVE: To examine the effects of the 2 nucleus pulposus (NP) cell populations, notochordal cells (NC) and chondrocyte-like cells (CC) on neurite outgrowth from DRGs. SUMMARY OF BACKGROUND DATA: NP consists of at least 2 cell populations, NC and CC. The cells in NP have been shown to be responsible for negative effects on neurite outgrowth in vitro and on nerve tissue in vivo. It is unknown whether 1 cell type or combinations of the 2 cell types are responsible for the reported effects. METHODS: A total of 939 DRGs from newborn Sprague Dawley rats were harvested and placed in culture dishes. After 24 hours, the neurite outgrowth was measured. NP was harvested from tail discs of adult rats and the NP cells were separated into 2 populations, NC and CC. The cell populations were applied to the DRG culture in different cell concentrations and combinations, and compared to medium. After 24 hours of exposure, the neurite outgrowth was reassessed and expressed as the ratio between the outgrowth at 48 and 24 hours culture. RESULTS: NC in intermediate and high concentration and CC in high concentration induced a significant inhibition of the neurite outgrowth compared to culture medium. Further, one of the combinations (low NC and high CC concentration) resulted in a significant inhibition of the neurite outgrowth. CONCLUSION: The present study demonstrated negative effects of NP cells on nerve tissue culture explants. The combination of low NC and high CC concentrations may mimic the situation in humans, where we have an increased proportion of chondrocyte-like cells with age. The results from this study may provide a biologic explanation for the large variation of symptoms in disc herniation patients despite similar mechanical influence on nerve tissue.

Identification of cell proliferation zones, progenitor cells and a potential stem cell niche in the intervertebral disc region: a study in four species.

STUDY DESIGN: Descriptive experimental study in 4 different mammals. OBJECTIVE: To investigate cell proliferation/regeneration and localize stem cells/progenitor cells within the intervertebral disc (IVD). SUMMARY OF BACKGROUND DATA: Disc degeneration (DD) is believed to play a major role in patients with chronic lumbar pain. Lately, biologic treatment options for DD have gained increasing interest. Normal regeneration processes within the IVD and have previously been sparsely described and therefore it is of great interest to increase the knowledge about these processes. Methods. Detection of cell proliferations zones and label-retaining cells were done by in vivo 5-bromo-2-deoxyuridine (BrdU) labeling in 18 rabbits, killed after 4, 6, 10, 14, 28, or 56 days. Results were visualized with immunohistochemistry and fluorescence/confocal microscopy. Localization of progenitor cell were further investigated by immunohistochemistry using antibodies towards Notch1, Delta4, Jagged1, C-KIT, KI67, and Stro-1 in normal IVD from rabbits (n = 3), rats (n = 2), minipigs (n = 2), and in human degenerated IVD (n = 4). Further, flowcytometry analysis using progenitor markers were performed on additional human IVD cells (n = 3). RESULTS: BrdU positive cells were found in comparable numbers at early and late time points in most regions of the anulus fibrosus (AF) and nucleus pulposus demonstrating slow ongoing cell proliferation. In the AF border to ligament zone (AFo) and the perichondrium region (P) a stem cell niche-like pattern was determined (a high number of BrdU positive cells at early time points vs. only a few label retaining cells at later time points). In normal and DD tissue from the 4 investigated species progenitor cell markers were detected. Conclusion. The IVD is a tissue with ongoing slow cell proliferation both in the AF and the nucleus pulposus. The stem cell niche pattern detected in AFo and P can be suggested to play a role for IVD morphology and function. These findings may be of importance for the development of biologic treatment strategies.

Autologous nucleus pulposus primes T cells to develop into interleukin-4-producing effector cells: an experimental study on the autoimmune properties of nucleus pulposus.

An autoimmune response to herniated nucleus pulposus has been proposed to constitute a pathophysiologic mechanism for inducing sciatica based on the fact that nucleus pulposus under normal conditions is excluded from the development of immunological tolerance. The manifestation of an autoimmune response comprises different steps starting with antigen capture, continuing with activation of T helper (T(H)) cells and ending with production of autoantibodies. Activated T(H) cells differentiate into either T(H)1 cells, predominately producing proinflammatory cytokines such as interferon gamma (IFNgamma) or a T(H)2 subset mainly producing anti-inflammatory cytokines such as interleukin-4 (IL-4). The aim of the present study was to examine if exposure of autologous nucleus pulposus (NP) to the immune system for 3 weeks is potent enough to prime T(H) cells to differentiate into T(H)2 cells. The study was performed in a pig model allowing the exposure of NP to the immune system. To assess the polarization of T(H) cells the intracellular production of IFNgamma and IL-4 was measured in T cells by using flow cytometry. The revealed predominant production of IL-4 together with low production of IFNgamma in T cells after NP exposure to the immune system indicates that nucleus pulposus may prime T(H) cells to develop into IL-4-producing T(H)2 cells after being exposed to the immune system, for example, in association with disc herniation.

The expression of NK cell inhibitory receptors on cytotoxic T cells in B-cell chronic lymphocytic leukaemia (B-CLL).

Immune surveillance of tumours is mediated by cytotoxic T cells (CTL) that recognise tumour antigen. Reduced reactivity of CTL towards tumour cells could thus lead to disease progression and loss of tumour control. In B-cell chronic lymphocytic leukaemia (B-CLL), the function of tumour-reactive CTL seems to correlate inversely to disease stage. Inhibitory NK cell receptors are known to suppress the CTL response upon interaction with major histocompatibility complex (MHC) class I and increased expression of such receptors on CTL may inhibit the anti-tumour response. So, the aim of this study was to investigate the expression of NK cell inhibitory receptors on CTL in B-CLL patients and if such expression correlated to disease stage. CD8+ T cells from B-CLL patients in Binet stage A (n = 26) and stage C (n = 14) and healthy controls (n = 14) were analysed for the expression of killer immunoglobulin-like receptors (KIR) CD158a (KIR2DL1), CD158b (KIR2DL2), CD158e (KIR3DL1) and the C-type lectin receptor CD94, by flow cytometry analysis. Patients with advanced disease (Binet stage C) had a significantly greater percentage of CTL expressing CD158b, CD158e and CD94 than patients with non-progressive disease (Binet stage A) and healthy controls. Stage C patients also had a significantly higher percentage of CTL expressing CD158a than stage A patients. No statistically significant differences were found between Binet A patients and healthy controls. Our results suggest that increased expression of KIR and CD94 on CTL in advanced stage B-CLL may potentially contribute to the impaired anti-tumour immune response in these patients.