Reduced Differentiation Capacity Research Articles

Secreted microvesicular miR-31 inhibits osteogenic differentiation of mesenchymal stem cells.

SummaryDamage to cells and tissues is one of the driving forces of aging and age‐related diseases. Various repair systems are in place to counteract this functional decline. In particular, the property of adult stem cells to self‐renew and differentiate is essential for tissue homeostasis and regeneration. However, their functionality declines with age (Rando, 2006). One organ that is notably affected by the reduced differentiation capacity of stem cells with age is the skeleton. Here, we found that circulating microvesicles impact on the osteogenic differentiation capacity of mesenchymal stem cells in a donor‐age‐dependent way. While searching for factors mediating the inhibitory effect of elderly derived microvesicles on osteogenesis, we identified miR‐31 as a crucial component. We demonstrated that miR‐31 is present at elevated levels in the plasma of elderly and of osteoporosis patients. As a potential source of its secretion, we identified senescent endothelial cells, which are known to increase during aging in vivo (Erusalimsky, 2009). Endothelial miR‐31 is secreted within senescent cell‐derived microvesicles and taken up by mesenchymal stem cells where it inhibits osteogenic differentiation by knocking down its target Frizzled‐3. Therefore, we suggest that microvesicular miR‐31 in the plasma of elderly might play a role in the pathogenesis of age‐related impaired bone formation and that miR‐31 might be a valuable plasma‐based biomarker for aging and for a systemic environment that does not favor cell‐based therapies whenever osteogenesis is a limiting factor.

MP24-14 RADIATION THERAPY ATTENUATES REGENERATION OF THE UROTHELIUM IN NOVEL CELL CULTURE RADIATION MODEL

You have accessJournal of UrologyInfections/Inflammation/Cystic Disease of the Genitourinary Tract: Kidney & Bladder I1 Apr 2016MP24-14 RADIATION THERAPY ATTENUATES REGENERATION OF THE UROTHELIUM IN NOVEL CELL CULTURE RADIATION MODEL Bernadette Zwaans, Sarah Bartolone, Peter Levanovich, Monica Liebert, Michael Chancellor, and Laura Lamb Bernadette ZwaansBernadette Zwaans More articles by this author , Sarah BartoloneSarah Bartolone More articles by this author , Peter LevanovichPeter Levanovich More articles by this author , Monica LiebertMonica Liebert More articles by this author , Michael ChancellorMichael Chancellor More articles by this author , and Laura LambLaura Lamb More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.770AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Radiation cystitis (RC) is a chronic inflammatory condition of the bladder that results from extensive exposure to radiation therapy for the treatment of pelvic malignancies. RC is a severely debilitating condition due to its symptoms (e.g. hematuria) and the lack of reliable treatment options. The underlying mechanism that contributes to RC development is not well understood. In this study, we aim to investigate the contribution of radiation-induced urothelial damage to the development of RC. We hypothesize that radiation exposure weakens the urothelial layer by reducing cellular proliferation, survival and differentiation ability of basal urothelial cells. Consequently, the damaged urothelium becomes more permeable to urine, which can cause irritation to the underlying tissue. METHODS Normal human basal urothelial (UR15) cells, developed by Jim Bassuk at the University of Washington, were used to study the effect of radiation therapy on the urothelium. UR15 cells, immortalized using HPV E6/E7, were cultured in serum-free keratinocyte media and differentiated in 10% DMEM. Cells were irradiated and the effect of radiation on cellular proliferation, survival, and differentiation was determined. RESULTS Exposure of undifferentiated UR15 cells to radiation treatment resulted in a dose-dependent decline in cellular proliferation that lasted beyond 2 weeks in culture. This decline in cellular proliferation was both due to an increase in apoptosis (Annexin V) as well as a decrease in the number of cell divisions. Additionally, irradiated UR15 cells had reduced differentiation capacity as measured by the formation of tight junctions (E-cadherin), a marker of urothelial differentiation. Ongoing studies are determining cell survival of differentiated urothelial cells as well as identifying changes to urothelial permeability in response to radiation. CONCLUSIONS Using a novel urothelial radiation model our studies concluded that radiation exposure to the bladder damages the urothelium, at least in part by reducing proliferation and differentiation of basal urothelial cells. Furthermore, a decrease in E-cadherin localized to the cell membrane suggests an increase in bladder permeability. This model may be advantages to investigate therapeutic options that may treat or prevent radiation cystitis. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e275 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Bernadette Zwaans More articles by this author Sarah Bartolone More articles by this author Peter Levanovich More articles by this author Monica Liebert More articles by this author Michael Chancellor More articles by this author Laura Lamb More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Differences in the Osteogenic Differentiation Capacity of Omental Adipose-Derived Stem Cells in Obese Patients With and Without Metabolic Syndrome

Multiple studies have suggested that the reduced differentiation capacity of multipotent adipose tissue-derived mesenchymal stem cells (ASCs) in obese subjects could compromise their use in cell therapy. Our aim was to assess the osteogenic potential of omental ASCs and to examine the status of the isolated CD34(negative)-enriched fraction of omental-derived ASCs from subjects with different metabolic profiles. Omental ASCs from normal-weight subjects and subjects with or without metabolic syndrome were isolated, and the osteogenic potential of omental ASCs was evaluated. Additionally, osteogenic and clonogenic potential, proliferation rate, mRNA expression levels of proteins involved in redox balance, and fibrotic proteins were examined in the CD34(negative)-enriched fraction of omental-derived ASCs. Both the omental ASCs and the CD34(negative)-enriched fraction of omental ASCs from subjects without metabolic syndrome have a greater osteogenic potential than those from subjects with metabolic syndrome. The alkaline phosphatase and osteonectin mRNA were negatively correlated with nicotinamide adenine dinucleotide phosphate oxidase-2 mRNA and the mRNA expression levels of the fibrotic proteins correlated positively with nicotinamide adenine dinucleotide phosphate oxidase-5 mRNA and the homeostasis model assessment. Although the population doubling time was significantly higher in subjects with a body mass index of 25 kg/m(2) or greater, only the CD34(negative)-enriched omental ASC fraction in the subjects with metabolic syndrome had a higher population doubling time than the normal-weight subjects. The osteogenic, clonogenic, fibrotic potential, and proliferation rate observed in vitro suggest that omental ASCs from subjects without metabolic syndrome are more suitable for therapeutic osteogenic applications than those from subjects with metabolic syndrome.

Peritoneal milky spots serve as a hypoxic niche and favor gastric cancer stem/progenitor cell peritoneal dissemination through hypoxia-inducible factor 1α.

Peritoneal dissemination is the most common cause of death in gastric cancer patients. The hypoxic microenvironment plays a major role in controlling the tumor stem cell phenotype and is associated with patients' prognosis through hypoxia-inducible factor-1α (HIF-1α), a key transcriptional factor that responds to hypoxic stimuli. During the peritoneal dissemination process, gastric cancer stem/progenitor cells (GCSPCs) are thought to enter into and maintained in peritoneal milky spots (PMSs), which have hypoxic microenvironments. However, the mechanism through which the hypoxic environment of PMSs regulated GCSPC maintenance is still poorly understood. Here, we investigated whether hypoxic PMSs were an ideal cancer stem cell niche suitable for GCSPC engraftment. We also evaluated the mechanisms through which the HIF-1α-mediated hypoxic microenvironment regulated GCSPC fate. We observed a positive correlation between HIF-1α expression and gastric cancer peritoneal dissemination (GCPD) in gastric cancer patients. Furthermore, the GCSPC population expanded in primary gastric cancer cells under hypoxic condition in vitro, and hypoxic GCSPCs showed enhanced self-renewal ability, but reduced differentiation capacity, mediated by HIF-1α. In an animal model, GCSPCs preferentially resided in the hypoxic zone of PMSs; moreover, when the hypoxic microenvironment in PMSs was destroyed, GCPD was significantly alleviated. In conclusion, our results demonstrated that PMSs served as a hypoxic niche and favored GCSPCs peritoneal dissemination through HIF-1α both in vitro and in vivo. These results provided new insights into the GCPD process and may lead to advancements in the clinical treatment of gastric cancer. Stem Cells 2014;32:3062–3074

Variable induction of PRDM1 and differentiation in chronic lymphocytic leukemia is associated with anergy

AbstractDespite antigen engagement and intact B-cell–receptor (BCR) signaling, chronic lymphocytic leukemia (CLL) cells fail to undergo terminal differentiation. We hypothesized that such failure may be due to anergy, as CLL cells exhibit variable levels of nonresponsiveness to surface IgM stimulation that is reversible in vitro. Moreover, anergy is associated with reduced differentiation capacity in normal B cells. We investigated responses of CLL cells to two potent differentiation-promoting agents, IL-21 and cytosine guanine dinucleotide-enriched oligo-deoxynucleotides. The induction of PR domain-containing protein 1 (PRDM1; also known as Blimp-1), a critical regulator of plasmacytic differentiation, by these agents was closely correlated but varied between individual cases, despite functionally intact IL-21 receptor– and Toll-like receptor 9–mediated signal transducer and activator of transcription 3, and nuclear factor-κB pathways. PRDM1 induction was inversely correlated with the extent of anergy as measured by the ability to mobilize intracellular Ca2+ following BCR crosslinking. PRDM1 responsiveness was associated with other markers of differentiation and proliferation but not with differences in apoptosis. The ability to induce PRDM1 did correlate with differential transcriptional and epigenetic regulation of the PRDM1 gene. These studies extend our understanding of CLL pathobiology, demonstrating that reduced differentiation capacity may be a consequence of anergy. Epigenetic drugs may offer possibilities to reactivate PRDM1 expression as part of novel differentiation therapy approaches.

Biological and Physicochemical Characterization of a Serum-and Xeno-Free Chemically Defined Cryopreservation Procedure for Adult Human Progenitor Cells

While therapeutic cell transplantations using progenitor cells are increasingly evolving towards phase I and II clinical trials and chemically defined cell culture is established, standardization in biobanking is still in the stage of infancy. In this study, the EU FP6-funded CRYSTAL (CRYo-banking of Stem cells for human Therapeutic AppLication) consortium aimed to validate novel Standard Operating Procedures (SOPs) to perform and validate xeno-free and chemically defined cryopreservation of human progenitor cells and to reduce the amount of the potentially toxic cryoprotectant additive (CPA) dimethyl sulfoxide (DMSO). To achieve this goal, three human adult progenitor and stem cell populations-umbilical cord blood (UCB)-derived erythroid cells (UCB-ECs), UCB-derived endothelial colony forming cells (UCB-ECFCs), and adipose tissue (AT)-derived mesenchymal stromal cells (AT-MSCs)-were cryopreserved in chemically defined medium supplemented with 10% or 5% DMSO. Cell recovery, cell repopulation, and functionality were evaluated postthaw in comparison to cryopreservation in standard fetal bovine serum (FBS)-containing freezing medium. Even with a reduction of the DMSO CPA to 5%, postthaw cell count and viability assays indicated no overall significant difference versus standard cryomedium. Additionally, to compare cellular morphology/membrane integrity and ice crystal formation during cryopreservation, multiphoton laser-scanning cryomicroscopy (cryo-MPLSM) and scanning electron microscopy (SEM) were used. Neither cryo-MPLSM nor SEM indicated differences in membrane integrity for the tested cell populations under various conditions. Moreover, no influence was observed on functional properties of the cells following cryopreservation in chemically defined freezing medium, except for UCB-ECs, which showed a significantly reduced differentiation capacity after cryopreservation in chemically defined medium supplemented with 5% DMSO. In summary, these results demonstrate the feasibility and robustness of standardized xeno-free cryopreservation of different human progenitor cells and encourage their use even more in the field of tissue-engineering and regenerative medicine.

Intrinsic and Microenvironmental Defects Are Involved in the Age-Related Changes of Lin−c-kit+Hematopoietic Progenitor Cells

The aim of this study was to evaluate through cross-transplantation models the effect of aging on the number of Lin(-)c-kit+ hematopoietic progenitor cells, on their ability to differentiate towards a lymphocyte phenotype, and on the role of the microenvironment in hematopoietic differentiation. The absolute number of purified Lin(-)c-kit+ cells from bone marrow was significantly lower in aged than in young mice. When transplanted in young recipients, Lin(-)c-kit+ hematopoietic progenitor cells from aged mice showed a reduced differentiation capacity in T cells and NK cells, compared to Lin(-)c-kit+ cells from young animals. The role of microenvironment in Lin(-)c-kit+ hematopoietic progenitor cells differentiation was evaluated by injecting young Lin(-)c-kit+ cells in young or aged recipients, the latter transplanted or not with a young thymus. In these conditions, the differentiation of Lin(-)c-kit+ cells from young mice in T and NK cells was less efficient in aged than in young recipients, independently of thymus grafting in aged recipients. In addition to quantitative defects qualitative alterations were also present in Lin(-)c-kit+ cells from aged mice, as evidenced by the fact that the injection of Lin(-)c-kit+ cells from aged donors in young recipients differentiated in CD4+ T cells that retained an interleukin-4 (IL-4) production in-between young and old control values. In conclusion, we have demonstrated that aging is associated with numerical and functional alterations of Lin(-)c-kit+ hematopoietic progenitor cells as well as with an altered microenvironment that is required for Lin(-)c-kit+ cells differentiation toward a lymphocyte phenotype.

A Novel Technique to Propagate Primary Human Preadipocytes without Loss of Differentiation Capacity

The study of human preadipocytes is hampered by the limited availability of adipose tissue and low yield of cell preparation. Proliferation of preadipocytes using common protocols, including fetal bovine serum (FBS), results in a markedly reduced differentiation capacity. Therefore, we were interested in developing an improved culture system that allows the proliferation of human preadipocytes without loss of differentiation capacity. Adipose tissue samples were taken from subjects undergoing elective abdominal surgery. Cells were seeded at various densities and cultured using different formulations of proliferation media including factors such as fibroblast growth factor-2 (basic fibroblast growth factor), epidermal growth factor, insulin, and FBS either alone or in combination. Cells were counted and induced to differentiate after confluence. After complete differentiation, cells were harvested, and glycerol-3-phosphate dehydrogenase activity was measured. Cells were subcultured for up to five passages. The proliferation medium with 4 growth factors (PM4), consisting of 2.5% FBS, 10 ng/mL epidermal growth factor, 1 ng/mL basic fibroblast growth factor, and 8.7 muM insulin, resulted in lower doubling times at all seeding densities tested (0.05 x 10(4) to 1.5 x 10(4)) compared with medium supplemented with 10% FBS. In contrast to cells in FBS medium, cells grown with PM4 medium retained full differentiation rate (glycerol-3-phosphate dehydrogenase activity, 493 +/- 215 vs. 41 +/- 17 mU/mg, p < 0.01). Differentiation capacity was fully retained at least for up to three passages in PM4 medium. The use of PM4 medium results in substantial proliferation of human preadipocytes with preserved differentiation capacity. This novel technique represents a valuable tool for the study of human adipose tissue development and function starting from small samples.

Reduced chondrogenic potential of adipose tissue derived stromal cells correlates with an altered TGFβ receptor and BMP profile and is overcome by BMP‐6

Recent interest has focused on mesenchymal stem cells (MSC) for tissue engineering and regenerative therapy of cartilage defects. MSC originating from adipose tissue (ATSC) are attractive as they are easily available and abundant. They have similar properties like bone marrow derived MSC (BMSC), except for a reduced chondrogenic potential under standard culture conditions driven by TGFbeta. Aim of this study was to search for possible differences explaining the reduced differentiation capacity of ATSC and to eliminate it by adaptation of induction protocols. Expanded MSC were analyzed for their growth factor and related receptor repertoire and ATSC spheroid cultures were supplemented with BMP-2,-4,-6,-7, TGFbeta, FGFa, FGFb, IGF-1, and PTHrP alone or in combination with TGFbeta. In contrast to BMSC, ATSC showed reduced expression of BMP-2, -4, and -6 mRNA and did not express TGFbeta-receptor-I protein. Consistent with this, increased concentrations of TGFbeta did not improve chondrogenesis of ATSC. BMP6 treatment induced TGFbeta-receptor-I expression and combined application of TGFbeta and BMP-6 eliminated the reduced chondrogenic potential of ATSC inducing a gene expression profile similar to differentiated BMSC. Like in BMSC, chondrogenesis of ATSC was associated with hypertrophy according to premature collagen Type X expression, upregulation of alkaline-phosphatase activity and in vivo calcification of spheroids after ectopic transplantation in SCID mice. In conclusion, a distinct BMP and TGFbeta-receptor repertoire may explain the reduced chondrogenic capacity of ATSC in vitro, which could be compensated by exogenous application of lacking factors. Further studies should now be directed to induce chondrogenesis in the absence of hypertrophy.