Silk Hydrogel Substrate Stress Relaxation Primes Mesenchymal Stem Cell Behavior in 2D.

Tissue-mimetic silk hydrogels are being explored for diverse healthcare applications, including stem cell delivery. However, the impact of stress relaxation of silk hydrogels on human mesenchymal stem cell (MSC) biology is poorly defined. The aim of this study was to fabricate silk hydrogels with tuned mechanical properties that allowed the regulation of MSC biology in two dimensions. The silk content and stiffness of both elastic and viscoelastic silk hydrogels were kept constant to permit direct comparisons. Gene expression of IL-1ß, IL-6, LIF, BMP-6, BMP-7, and protein tyrosine phosphatase receptor type C were substantially higher in MSCs cultured on elastic hydrogels than those on viscoelastic hydrogels, whereas this pattern was reversed for insulin, HNF-1A, and SOX-2. Protein expression was also mechanosensitive and the elastic cultures showed strong activation of IL-1ß signaling in response to hydrogel mechanics. An elastic substrate also induced higher consumption of glucose and aspartate, coupled with a higher secretion of lactate, than was observed in MSCs grown on viscoelastic substrate. However, both silk hydrogels changed the magnitude of consumption of glucose, pyruvate, glutamine, and aspartate, and also metabolite secretion, resulting in an overall lower metabolic activity than that found in control cells. Together, these findings describe how stress relaxation impacts the overall biology of MSCs cultured on silk hydrogels.

JNK signaling prevents biliary cyst formation through a CASPASE-8-dependent function of RIPK1 during aging.

The c-Jun N-terminal kinase (JNK) signaling pathway mediates adaptation to stress signals and has been associated with cell death, cell proliferation, and malignant transformation in the liver. However, up to now, its function was experimentally studied mainly in young mice. By generating mice with combined conditional ablation of Jnk1 and Jnk2 in liver parenchymal cells (LPCs) (JNK1/2LPC-KO mice; KO, knockout), we unraveled a function of the JNK pathway in the regulation of liver homeostasis during aging. Aging JNK1/2LPC-KO mice spontaneously developed large biliary cysts that originated from the biliary cell compartment. Mechanistically, we could show that cyst formation in livers of JNK1/2LPC-KO mice was dependent on receptor-interacting protein kinase 1 (RIPK1), a known regulator of cell survival, apoptosis, and necroptosis. In line with this, we showed that RIPK1 was overexpressed in the human cyst epithelium of a subset of patients with polycystic liver disease. Collectively, these data reveal a functional interaction between JNK signaling and RIPK1 in age-related progressive cyst development. Thus, they provide a functional linkage between stress adaptation and programmed cell death (PCD) in the maintenance of liver homeostasis during aging.

[Screening for Anxiety in People with an Intellectual Disability: German Version of the "Glasgow Anxiety Scale for People with an Intellectual Disability" (GAS-ID)]. / Screeninginstrument für Angst bei Menschen mit Intelligenzminderung: Deutsche Version der "Glasgow Anxiety Scale for people with an Intellectual Disability" (GAS-ID).

OBJECTIVE: As of yet, there exists no German-language self-rating questionnaire as a screening for anxiety disorders in people with an intellectual disability. Therefore the Glasgow Anxiety Scale for people with an Intellectual Disability (GAS-ID) was translated into German and its psychometric properties were assessed. METHODS: Internal consistency and concurrent validity were tested in 34 adults with learning difficulties and mild and moderate intellectual disability. Convergent validity was estimated by using data from a clinical sample of 38 persons without intellectual disability. RESULTS: The GAS-ID discriminates between intellectually disabled subjects with an anxiety disorder and subjects without comorbid mental disorders resulting in a sensitivity of 100 % and a specificity of 87 %. It also demonstrates a very good internal consistency (Cronbachs α = 0.90) and a high convergent validity (with BAI: r = 0.76). CONCLUSION: The German GAS-ID is a reliable, valid and economically applicable self-report screening instrument for assessing anxiety in people with intellectual disability. However, minor revision of items and future research is needed to investigate the validity of the GAS-ID.

Comprehensive characterization of chorionic villi-derived mesenchymal stromal cells from human placenta.

BACKGROUND: Studies in which mesenchymal stromal cells (MSC) from the placenta are compared with multiple MSC types from other sources are rare. The chorionic plate of the human placenta is mainly composed of fetal blood vessels embedded in fetal stroma tissue, lined by trophoblastic cells and organized into chorionic villi (CV) structures. METHODS: We comprehensively characterized human MSC collected from postnatal human chorionic villi of placenta (CV-MSC) by analyzing their growth and proliferation potential, differentiation, immunophenotype, extracellular matrix production, telomere length, aging phenotype, and plasticity. RESULTS: Immunophenotypic characterization of CV-MSC confirmed the typical MSC marker expression as defined by the International Society for Cellular Therapy. The surface marker profile was consistent with increased potential for proliferation, vascular localization, and early myogenic marker expression. CV-MSC retained multilineage differentiation potential and extracellular matrix remodeling properties. They have undergone reduced telomere loss and delayed onset of cellular senescence as they aged in vitro compared to three other MSC sources. We present evidence that increased human telomerase reverse transcriptase gene expression could not explain the exceptional telomere maintenance and senescence onset delay in cultured CV-MSC. Our in-vitro tumorigenesis detection assay suggests that CV-MSC are not prone to undergo malignant transformation during long-term in-vitro culture. Besides SOX2 expression, no other pluripotency features were observed in early and late passages of CV-MSC. CONCLUSIONS: Our work brings forward two remarkable characteristics of CV-MSC, the first being their extended life span as a result of delayed replicative senescence and the second being a delayed aged phenotype characterized by improved telomere length maintenance. MSC from human placenta are very attractive candidates for stem cell-based therapy applications.

The radical SAM protein HemW is a heme chaperone.

Radical S-adenosylmethionine (SAM) enzymes exist in organisms from all kingdoms of life, and all of these proteins generate an adenosyl radical via the homolytic cleavage of the S-C(5') bond of SAM. Of particular interest are radical SAM enzymes, such as heme chaperones, that insert heme into respiratory enzymes. For example, heme chaperones insert heme into target proteins but have been studied only for the formation of cytochrome c-type hemoproteins. Here, we report that a radical SAM protein, the heme chaperone HemW from bacteria, is required for the insertion of heme b into respiratory chain enzymes. As other radical SAM proteins, HemW contains three cysteines and one SAM coordinating an [4Fe-4S] cluster, and we observed one heme per subunit of HemW. We found that an intact iron-sulfur cluster was required for HemW dimerization and HemW-catalyzed heme transfer but not for stable heme binding. A bacterial two-hybrid system screen identified bacterioferritins and the heme-containing subunit NarI of the respiratory nitrate reductase NarGHI as proteins that interact with HemW. We also noted that the bacterioferritins potentially serve as heme donors for HemW. Of note, heme that was covalently bound to HemW was actively transferred to a heme-depleted, catalytically inactive nitrate reductase, restoring its nitrate-reducing enzyme activity. Finally, the human HemW orthologue radical SAM domain-containing 1 (RSAD1) stably bound heme. In conclusion, our findings indicate that the radical SAM protein family HemW/RSAD1 is a heme chaperone catalyzing the insertion of heme into hemoproteins.

Zebrafish In-Vivo Screening for Compounds Amplifying Hematopoietic Stem and Progenitor Cells: - Preclinical Validation in Human CD34+ Stem and Progenitor Cells.

The identification of small molecules that either increase the number and/or enhance the activity of human hematopoietic stem and progenitor cells (hHSPCs) during ex vivo expansion remains challenging. We used an unbiased in vivo chemical screen in a transgenic (c-myb:EGFP) zebrafish embryo model and identified histone deacetylase inhibitors (HDACIs), particularly valproic acid (VPA), as significant enhancers of the number of phenotypic HSPCs, both in vivo and during ex vivo expansion. The long-term functionality of these expanded hHSPCs was verified in a xenotransplantation model with NSG mice. Interestingly, VPA increased CD34+ cell adhesion to primary mesenchymal stromal cells and reduced their in vitro chemokine-mediated migration capacity. In line with this, VPA-treated human CD34+ cells showed reduced homing and early engraftment in a xenograft transplant model, but retained their long-term engraftment potential in vivo, and maintained their differentiation ability both in vitro and in vivo. In summary, our data demonstrate that certain HDACIs lead to a net expansion of hHSPCs with retained long-term engraftment potential and could be further explored as candidate compounds to amplify ex-vivo engineered peripheral blood stem cells.

Bone marrow niche-mimetics modulate HSPC function via integrin signaling.

The bone marrow (BM) microenvironment provides critical physical cues for hematopoietic stem and progenitor cell (HSPC) maintenance and fate decision mediated by cell-matrix interactions. However, the mechanisms underlying matrix communication and signal transduction are less well understood. Contrary, stem cell culture is mainly facilitated in suspension cultures. Here, we used bone marrow-mimetic decellularized extracellular matrix (ECM) scaffolds derived from mesenchymal stromal cells (MSCs) to study HSPC-ECM interaction. Seeding freshly isolated HSPCs adherent (AT) and non-adherent (SN) cells were found. We detected enhanced expansion and active migration of AT-cells mediated by ECM incorporated stromal derived factor one. Probing cell mechanics, AT-cells displayed naïve cell deformation compared to SN-cells indicating physical recognition of ECM material properties by focal adhesion. Integrin αIIb (CD41), αV (CD51) and ß3 (CD61) were found to be induced. Signaling focal contacts via ITGß3 were identified to facilitate cell adhesion, migration and mediate ECM-physical cues to modulate HSPC function.

Combined influence of biophysical and biochemical cues on maintenance and proliferation of hematopoietic stem cells.

Homeostasis of hematopoietic stem and progenitor cells (HSPC) is controlled by a combination of biochemical and biophysical environmental cues in the bone marrow (BM) niche, where a tight balance of quiescence and proliferation of HSPC is maintained. Specifically, alongside soluble factors and extracellular matrix (ECM) proteins, spatial confinement and ECM stiffness have been recognized to be critical for regulation of HSPC fate. Here we employ a modular, glycosaminoglycan (GAG)-based biohybrid hydrogel system to balance proliferation of human HSPC and maintenance of quiescent hematopoietic stem cells (HSC) through simultaneous regulation of exogenous biochemical and biophysical cues. Our results demonstrate that HSPC respond to increased spatial confinement with lowered proliferation and cell cycling, which results in higher frequency of quiescent LTC-IC (long-term culture initiating cells), while GAG-rich 3D environments further support maintenance of the cells.

Human Bone Marrow Stromal Cells: A Reliable, Challenging Tool for In Vitro Osteogenesis and Bone Tissue Engineering Approaches.

Adult human bone marrow stromal cells (hBMSC) are important for many scientific purposes because of their multipotency, availability, and relatively easy handling. They are frequently used to study osteogenesis in vitro. Most commonly, hBMSC are isolated from bone marrow aspirates collected in clinical routine and cultured under the "aspect plastic adherence" without any further selection. Owing to the random donor population, they show a broad heterogeneity. Here, the osteogenic differentiation potential of 531 hBMSC was analyzed. The data were supplied to correlation analysis involving donor age, gender, and body mass index. hBMSC preparations were characterized as follows: (a) how many passages the osteogenic characteristics are stable in and (b) the influence of supplements and culture duration on osteogenic parameters (tissue nonspecific alkaline phosphatase (TNAP), octamer binding transcription factor 4, core-binding factor alpha-1, parathyroid hormone receptor, bone gla protein, and peroxisome proliferator-activated protein γ). The results show that no strong prediction could be made from donor data to the osteogenic differentiation potential; only the ratio of induced TNAP to endogenous TNAP could be a reliable criterion. The results give evidence that hBMSC cultures are stable until passage 7 without substantial loss of differentiation potential and that established differentiation protocols lead to osteoblast-like cells but not to fully authentic osteoblasts.

Do breast cups improve breast cancer dosimetry? A comparative study for patients with large or pendulous breasts.

PURPOSE: Treating patients with large or pendulous breasts is challenging. Although brassiere cups are currently in use, no study has yet been carried out to assess their dosimetric impact. The aim of the present study was to evaluate the possible dosimetric advantages of the use of breast cups on patients with large or pendulous breasts. MATERIALS AND METHODS: Two CT studies were carried out on 12 breast cancer patients with large or pendulous breasts, with one study involving the use of breast cups. Radiation plans were developed in accordance with each of the CT studies. The following were compared: planning target volume (PTV), volume irradiated by the 95% isodose, conformity index, homogeneity index, mean lung dose, and mean heart dose was also compared for left breast treatment. The plan involving the use of cups was found to be the best option, leading to all patients being treated with cups. The resulting acute toxicity and cosmesis were also recorded. Both scenarios involved the use of film dosimetry to evaluate the skin doses. RESULTS: The use of breast cups resulted in a significant reduction of the PTV volume (from 1640 cm3 to 1283 cm3), of the irradiated volume (from 2154 cm3 to 1477 cm3) and of the conformity index (from 1383 to 1213). Despite slight improvements in the homogeneity index (from 0.12 to 0.10), statistical significance was not attained. The use of breast cups also led to significant dose reductions in V20 for lung (from 13.7% to 1.7%) and V5 for heart (from 9.8% to 2.7%). No differences in acute toxicity or cosmesis were observed compared to patients treated without cups. CONCLUSIONS: Our results show that the use of brassiere cups during breast radiation therapy leads to improvements in the main dosimetric factors analyzed. Furthermore, modifications to standard irradiation protocols are not required. In summary, we consider the technique of using breast cups with radiation therapy highly appropriate when treating breast cancer patients with large or pendulous breasts.

An optimized posterior axillary boost technique in radiation therapy to supraclavicular and axillary lymph nodes: a comparative study.

To assess the advantages of an optimized posterior axillary (AX) boost technique for the irradiation of supraclavicular (SC) and AX lymph nodes. Five techniques for the treatment of SC and levels I, II, and III AX lymph nodes were evaluated for 10 patients selected at random: a direct anterior field (AP); an anterior to posterior parallel pair (AP-PA); an anterior field with a posterior axillary boost (PAB); an anterior field with an anterior axillary boost (AAB); and an optimized PAB technique (OptPAB). The target coverage, hot spots, irradiated volume, and dose to organs at risk were evaluated and a statistical analysis comparison was performed. The AP technique delivered insufficient dose to the deeper AX nodes. The AP-PA technique produced larger irradiated volumes and higher mean lung doses than the other techniques. The PAB and AAB techniques originated excessive hot spots in most of the cases. The OptPAB technique produced moderate hot spots while maintaining a similar planning target volume (PTV) coverage, irradiated volume, and dose to organs at risk. This optimized technique combines the advantages of the PAB and AP-PA techniques, with moderate hot spots, sufficient target coverage, and adequate sparing of normal tissues. The presented technique is simple, fast, and easy to implement in routine clinical practice and is superior to the techniques historically used for the treatment of SC and AX lymph nodes.

Tightly anchored tissue-mimetic matrices as instructive stem cell microenvironments.

A major obstacle in defining the exact role of extracellular matrix (ECM) in stem cell niches is the lack of suitable in vitro methods that recapitulate complex ECM microenvironments. Here we describe a methodology that permits reliable anchorage of native cell-secreted ECM to culture carriers. We validated our approach by fabricating two types of human bone marrow-specific ECM substrates that were robust enough to support human mesenchymal stem cells (MSCs) and hematopoietic stem and progenitor cells in vitro. We characterized the molecular composition, structural features and nanomechanical properties of the MSC-derived ECM preparations and demonstrated their ability to support expansion and differentiation of bone marrow stem cells. Our methodology enables the deciphering and modulation of native-like multicomponent ECMs of tissue-resident stem cells and will therefore prepare the ground for a more rational design of engineered stem cell niches.

Differential effects of mixed lymphocyte reaction supernatant on human mesenchymal stromal cells.

The concept that mesenchymal stromal cells (MSCs), a component of the hematopoietic microenvironment, can be a target for alloreactive effector cells in the context of graft-vs-host disease has not been investigated in detail. Mixed lymphocyte reaction (MLR) supernatant was used to mimic the inflammatory milieu induced by an allogeneic immune response in vitro. In addition to phenotype and proliferation, we monitored MSC differentiation, gene expression, and support of CD34(+) hematopoietic stem and progenitor cells after priming with MLR supernatant. Priming of MSCs with MLR supernatant led to an 11-fold decrease in cobblestone area-forming cells in the 4-week coculture (p < 0.05) and a threefold decrease of colony-forming unit macrophage in the colony-forming cell assay (p < 0.05). MSC proliferation over 8 days was increased 2.5-fold (p < 0.05). Osteogenic differentiation was enhanced, while adipogenesis was concurrently suppressed. In addition, the surface expression of HLA-DR and intercellular adhesion molecule-1 was increased 20-fold (p = 0.06) and 45-fold (p < 0.05), respectively. This was associated with increased adhesion of hematopoietic stem and progenitor cells to MLR-treated MSCs. In summary, our data shed light on the dysfunction of the stromal environment during graft-vs-host disease, possibly aggravating cytopenia and leading to an enhanced immunogenicity of MSCs.

A selective inhibitor reveals PI3Kγ dependence of T(H)17 cell differentiation.

We devised a high-throughput chemoproteomics method that enabled multiplexed screening of 16,000 compounds against native protein and lipid kinases in cell extracts. Optimization of one chemical series resulted in CZC24832, which is to our knowledge the first selective inhibitor of phosphoinositide 3-kinase γ (PI3Kγ) with efficacy in in vitro and in vivo models of inflammation. Extensive target- and cell-based profiling of CZC24832 revealed regulation of interleukin-17-producing T helper cell (T(H)17) differentiation by PI3Kγ, thus reinforcing selective inhibition of PI3Kγ as a potential treatment for inflammatory and autoimmune diseases.

Predicting compliance and survival in palliative whole-brain radiotherapy for brain metastases.

OBJECTIVE: Brain radiotherapy is the main treatment for patients with brain metastases but its goal is just symptom control. Our aim was to study if different performance tools, used in geriatric practice, could improve patient selection for decision-making in the palliative brain radiotherapy setting. PATIENTS AND METHODS: Data from 61 consecutive patients were analysed. In addition to Karnofsky Performance Status (KPS) their physical activity was assessed by means of the activity of daily living (ADL) and instrumental ADL (IADL) scales. A neurocognitive evaluation was performed with the Pfeiffer Short Portable Mental Status Questionnaire (SPMSQ) and with the Mini-Mental Status Exam (MMSE). Radiotherapy compliance and short survival were the endpoints of the study. RESULTS: High rates of cognitive impairment were found by both neurocognitive tools (Pfeiffer: 19.7% of patients; MMSE: 30%). Dependence was also highly prevalent, either measured by the ADL (50.8%) or by the IADL (43.3%). Nearly one third (27.9%) of patients died soon after radiotherapy evaluation. Longer survival was related to female, younger than 60 years, breast cancer primary tumour, steroid response, RPA class, and higher performance and neurocognitive score tools. A premature death was associated with neurocognitive tools, IADL and longer interval from brain metastatic diagnosis to radiotherapy. Twenty-three percent of patients were not able to finish the WBRT course due to clinical deterioration. The only variable related to compliance was a low MMSE score. CONCLUSIONS: Results suggest that the geriatric tools analysed could offer information on brain palliative radiotherapy complementary to that offered by the more usual tools. It will be interesting to study if our data could be extrapolated to the general palliative oncological field.

Hematopoietic stem cells in co-culture with mesenchymal stromal cells--modeling the niche compartments in vitro.

BACKGROUND: Hematopoietic stem cells located in the bone marrow interact with a specific microenvironment referred to as the stem cell niche. Data derived from ex vivo co-culture systems using mesenchymal stromal cells as a feeder cell layer suggest that cell-to-cell contact has a significant impact on the expansion, migratory potential and 'stemness' of hematopoietic stem cells. Here we investigated in detail the spatial relationship between hematopoietic stem cells and mesenchymal stromal cells during ex vivo expansion. DESIGN AND METHODS: In the co-culture system, we defined three distinct localizations of hematopoietic stem cells relative to the mesenchymal stromal cell layer: (i) those in supernatant (non-adherent cells); (ii) those adhering to the surface of mesenchymal stromal cells (phase-bright cells) and (iii) those beneath the mesenchymal stromal cells (phase-dim cells). Cell cycle, proliferation, cell division and immunophenotype of these three cell fractions were evaluated from day 1 to 7. RESULTS: Phase-bright cells contained the highest proportion of cycling progenitors during co-culture. In contrast, phase-dim cells divided much more slowly and retained a more immature phenotype compared to the other cell fractions. The phase-dim compartment was soon enriched for CD34(+)/CD38(-) cells. Migration beneath the mesenchymal stromal cell layer could be hampered by inhibiting integrin beta1 or CXCR4. CONCLUSIONS: Our data suggest that the mesenchymal stromal cell surface is the predominant site of proliferation of hematopoietic stem cells, whereas the compartment beneath the mesenchymal stromal cell layer seems to mimic the stem cell niche for more immature cells. The SDF-1/CXCR4 interaction and integrin-mediated cell adhesion play important roles in the distribution of hematopoietic stem cells in the co-culture system.

Impact of CXCR4 inhibition on FLT3-ITD-positive human AML blasts.

OBJECTIVE: Internal tandem duplication (ITD) mutations of the FLT3 receptor are associated with a high incidence of relapse in acute myeloid leukemia (AML). Expression of the CXCR4 receptor in FLT3-ITD-positive AML is correlated with poor outcome, and inhibition of CXCR4 was shown to sensitize AML blasts toward chemotherapy. The aim of this study was to evaluate the impact of FLT3-ITD on cell proliferation and CXCR4-dependent migration in human hematopoietic progenitor cells and to investigate their response to CXCR4 inhibition. MATERIALS AND METHODS: We used primary blasts from patients with FLT3-ITD or FLT3 wild-type AML. In addition, human CD34(+) hematopoietic progenitor cells were transduced to >70% with retroviral vectors containing human FLT3-ITD. RESULTS: We found that FLT3-ITD transgene overexpressing human hematopoietic progenitor cells show strongly reduced migration toward stromal-derived factor-1 in vitro and display significantly reduced bone marrow homing in nonobese diabetic severe combined immunodeficient mice. Cocultivation of FLT3-ITD-positive AML blasts or hematopoietic progenitor cells on bone marrow stromal cells resulted in a strong proliferation advantage and increased early cobblestone area-forming cells compared to FLT3-wild-type AML blasts. Addition of the CXCR4 inhibitor AMD3100 to the coculture significantly reduced both cobblestone area-forming cells and proliferation of FLT3-ITD-positive cells, but did not affect FLT3-wild-type cells-highlighting the critical interaction between CXCR4 and FLT3-ITD. CONCLUSION: CXCR4 inhibition to decrease cell proliferation and to control the leukemic burden may provide a novel therapeutic strategy in patients with advanced FLT3-ITD-positive AML.

Engineered extracellular matrices modulate the expression profile and feeder properties of bone marrow-derived human multipotent mesenchymal stromal cells.

The bone marrow harbors multipotent mesenchymal stromal cells (MSCs) that nurture hematopoietic stem cells (HSCs). The extracellular matrix (ECM) is an integral part of the bone marrow, and the aim of this study was therefore to examine the effect of engineered ECM substrates on MSC gene expression over time and to determine quantitatively the functional ability of ECM-cultured MSCs to support HSCs. ECMs were surface immobilized using thin films of maleic anhydride to covalently immobilize tropocollagen or fibrillar collagen type I to the substrate. Where indicated, collagen type I fibrils were supplemented with heparin or hyaluronic acid. All surfaces maintained MSC viability and supported cell expansion. Microarray analysis of MSCs cultured on engineered ECM substrates revealed that culture time, as well as substrate composition, significantly affected expression levels. Based on these studies, it was possible to predict the effect of these substrates on in vitro HSC clonogenicity and self-renewal. The ability to regulate the expression of stromal factors using engineered ECM is exciting and warrants further studies to identify the ECM components and combinations that maximize the expansion of clonogenic HSCs.

Direct contact with mesenchymal stromal cells affects migratory behavior and gene expression profile of CD133+ hematopoietic stem cells during ex vivo expansion.

OBJECTIVE: To investigate the impact of direct contact between mesenchymal stromal cells (MSCs) and CD133(+) hematopoietic stem cells in terms of expansion potential, differentiation, migratory capacity, and gene expression profile. MATERIALS AND METHODS: CD133(+)-purified hematopoietic progenitor cells were cultured for 7 days on subconfluent MSCs supplemented with growth-factor-containing medium. After ex vivo expansion, nonadherent and adherent cells were collected and analyzed separately. RESULTS: The adherent cell population was less differentiated than the nonadherent fraction. CXCR4 was upregulated in the adherent fraction, which was associated with a higher migration capacity toward a stromal cell-derived factor-1 gradient. Colony-forming unit granulocyte-macrophage and long-term culture-initiation cell assays demonstrated a higher clonogenicity and repopulating capacity of the adherent fraction. Genes involved in adhesion, cell-cycle control, motility, and self-renewal were more highly expressed in the adherent fraction. CONCLUSION: Adhesion and direct cell-to-cell contact with an MSC feeder layer supports ex vivo expansion, migratory potential, and stemness of CD133(+) hematopoietic progenitor cells.

Enumeration of functionally active anti-Aspergillus T-cells in human peripheral blood.

Invasive aspergillosis remains a life-threatening complication in patients undergoing allogeneic stem cell transplantation (SCT). Since CD4(+) T-cells provide a critical secondary defense against Aspergillus spp., the quantification of "functional" anti-Aspergillus T-cells might be important in the clinical care of allogeneic transplant patients. We present a rapid, simple and reproducible method to enumerate functionally active, cytokine-producing anti-Aspergillus T-cells in peripheral blood by means of flow cytometry, by which these cells were also phenotypically characterized as memory CD4(+) T-cells. When using 100,000 PBMCs and requiring a minimum of 50 events, at least one anti-Aspergillus T-cell among 1000 CD4(+) T-cells can be detected. Compared to healthy individuals, the number of anti-Aspergillus T-cells in patients up to one year after SCT was significantly lower. The presented method might help to define hematopoietic transplant recipients who will benefit from adoptive transfer of anti-Aspergillus T cells.