Tenascin-C is required for normal Wnt/β-catenin signaling in the whisker follicle stem cell niche.

Whisker follicles have multiple stem cell niches, including epidermal stem cells in the bulge as well as neural crest-derived stem cells and mast cell progenitors in the trabecular region. The neural crest-derived stem cells are a pool of melanocyte precursors. Previously, we found that the extracellular matrix glycoproteins tenascin-C and tenascin-W are expressed near CD34-positive cells in the trabecular stem cell niche of mouse whisker follicles. Here, we analyzed whiskers from tenascin-C knockout mice and found intrafollicular adipocytes and supernumerary mast cells. As Wnt/β-catenin signaling promotes melanogenesis and suppresses the differentiation of adipocytes and mast cells, we analyzed β-catenin subcellular localization in the trabecular niche. We found cytoplasmic and nuclear β-catenin in wild-type mice reflecting active Wnt/β-catenin signaling, whereas β-catenin in tenascin-C knockout mice was mostly cell membrane-associated and thus transcriptionally inactive. Furthermore, cells expressing the Wnt/β-catenin target gene cyclin D1 were enriched in the CD34-positive niches of wild-type compared to tenascin-C knockout mice. We then tested the effects of tenascins on this signaling pathway. We found that tenascin-C and tenascin-W can be co-precipitated with Wnt3a. In vitro, substrate bound tenascins promoted β-catenin-mediated transcription in the presence of Wnt3a, presumably due to the sequestration and concentration of Wnt3a near the cell surface. We conclude that the presence of tenascin-C in whiskers assures active Wnt/β-catenin signaling in the niche thereby maintaining the stem cell pool and suppressing aberrant differentiation, while in the knockout mice with reduced Wnt/β-catenin signaling, stem cells from the trabecular niche can differentiate into ectopic adipocytes and mast cells.

Leukemic stem cell frequency: a strong biomarker for clinical outcome in acute myeloid leukemia.

INTRODUCTION Treatment failure in acute myeloid leukemia is probably caused by the presence of leukemia initiating cells, also referred to as leukemic stem cells, at diagnosis and their persistence after therapy. Specific identification of leukemia stem cells and their discrimination from normal hematopoietic stem cells would greatly contribute to risk stratification and could predict possible relapses. RESULTS For identification of leukemic stem cells, we developed flow cytometric methods using leukemic stem cell associated markers and newly-defined (light scatter) aberrancies. The nature of the putative leukemic stem cells and normal hematopoietic stem cells, present in the same patient's bone marrow, was demonstrated in eight patients by the presence or absence of molecular aberrancies and/or leukemic engraftment in NOD-SCID IL-2Rγ-/- mice. At diagnosis (n=88), the frequency of the thus defined neoplastic part of CD34+CD38- putative stem cell compartment had a strong prognostic impact, while the neoplastic parts of the CD34+CD38+ and CD34- putative stem cell compartments had no prognostic impact at all. After different courses of therapy, higher percentages of neoplastic CD34+CD38- cells in complete remission strongly correlated with shorter patient survival (n=91). Moreover, combining neoplastic CD34+CD38- frequencies with frequencies of minimal residual disease cells (n=91), which reflect the total neoplastic burden, revealed four patient groups with different survival. CONCLUSION AND PERSPECTIVE Discrimination between putative leukemia stem cells and normal hematopoietic stem cells in this large-scale study allowed to demonstrate the clinical importance of putative CD34+CD38- leukemia stem cells in AML. Moreover, it offers new opportunities for the development of therapies directed against leukemia stem cells, that would spare normal hematopoietic stem cells, and, moreover, enables in vivo and ex vivo screening for potential efficacy and toxicity of new therapies.

The molecular signature of the stroma response in prostate cancer-induced osteoblastic bone metastasis highlights expansion of hematopoietic and prostate epithelial stem cell niches

The reciprocal interaction between cancer cells and the tissue-specific stroma is critical for primary and metastatic tumor growth progression. Prostate cancer cells colonize preferentially bone (osteotropism), where they alter the physiological balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption, and elicit prevalently an osteoblastic response (osteoinduction). The molecular cues provided by osteoblasts for the survival and growth of bone metastatic prostate cancer cells are largely unknown. We exploited the sufficient divergence between human and mouse RNA sequences together with redefinition of highly species-specific gene arrays by computer-aided and experimental exclusion of cross-hybridizing oligonucleotide probes. This strategy allowed the dissection of the stroma (mouse) from the cancer cell (human) transcriptome in bone metastasis xenograft models of human osteoinductive prostate cancer cells (VCaP and C4-2B). As a result, we generated the osteoblastic bone metastasis-associated stroma transcriptome (OB-BMST). Subtraction of genes shared by inflammation, wound healing and desmoplastic responses, and by the tissue type-independent stroma responses to a variety of non-osteotropic and osteotropic primary cancers generated a curated gene signature ("Core" OB-BMST) putatively representing the bone marrow/bone-specific stroma response to prostate cancer-induced, osteoblastic bone metastasis. The expression pattern of three representative Core OB-BMST genes (PTN, EPHA3 and FSCN1) seems to confirm the bone specificity of this response. A robust induction of genes involved in osteogenesis and angiogenesis dominates both the OB-BMST and Core OB-BMST. This translates in an amplification of hematopoietic and, remarkably, prostate epithelial stem cell niche components that may function as a self-reinforcing bone metastatic niche providing a growth support specific for osteoinductive prostate cancer cells. The induction of this combinatorial stem cell niche is a novel mechanism that may also explain cancer cell osteotropism and local interference with hematopoiesis (myelophthisis). Accordingly, these stem cell niche components may represent innovative therapeutic targets and/or serum biomarkers in osteoblastic bone metastasis.

A mouse model of HIES reveals pro- and anti-inflammatory functions of STAT3.

Mutations of STAT3 underlie the autosomal dominant form of hyperimmunoglobulin E syndrome (HIES). STAT3 has critical roles in immune cells and thus, hematopoietic stem cell transplantation (HSCT), might be a reasonable therapeutic strategy in this disease. However, STAT3 also has critical functions in nonhematopoietic cells and dissecting the protean roles of STAT3 is limited by the lethality associated with germline deletion of Stat3. Thus, predicting the efficacy of HSCT for HIES is difficult. To begin to dissect the importance of STAT3 in hematopoietic and nonhematopoietic cells as it relates to HIES, we generated a mouse model of this disease. We found that these transgenic mice recapitulate multiple aspects of HIES, including elevated serum IgE and failure to generate Th17 cells. We found that these mice were susceptible to bacterial infection that was partially corrected by HSCT using wild-type bone marrow, emphasizing the role played by the epithelium in the pathophysiology of HIES.

The transcriptome of equine peripheral blood mononuclear cells.

Complete transcriptomic data at high resolution are available only for a few model organisms with medical importance. The gene structures of non-model organisms are mostly computationally predicted based on comparative genomics with other species. As a result, more than half of the horse gene models are known only by projection. Experimental data supporting these gene models are scarce. Moreover, most of the annotated equine genes are single-transcript genes. Utilizing RNA sequencing (RNA-seq) the experimental validation of predicted transcriptomes has become accessible at reasonable costs. To improve the horse genome annotation we performed RNA-seq on 561 samples of peripheral blood mononuclear cells (PBMCs) derived from 85 Warmblood horses. The mapped sequencing reads were used to build a new transcriptome assembly. The new assembly revealed many alternative isoforms associated to known genes or to those predicted by the Ensembl and/or Gnomon pipelines. We also identified 7,531 transcripts not associated with any horse gene annotated in public databases. Of these, 3,280 transcripts did not have a homologous match to any sequence deposited in the NCBI EST database suggesting horse specificity. The unknown transcripts were categorized as coding and noncoding based on predicted coding potential scores. Among them 230 transcripts had high coding potential score, at least 2 exons, and an open reading frame of at least 300 nt. We experimentally validated 9 new equine coding transcripts using RT-PCR and Sanger sequencing. Our results provide valuable detailed information on many transcripts yet to be annotated in the horse genome.

Slow conduction in mixed cultured strands of primary ventricular cells and stem cell-derived cardiomyocytes

Modern concepts for the treatment of myocardial diseases focus on novel cell therapeutic strategies involving stem cell-derived cardiomyocytes (SCMs). However, functional integration of SCMs requires similar electrophysiological properties as primary cardiomyocytes (PCMs) and the ability to establish intercellular connections with host myocytes in order to contribute to the electrical and mechanical activity of the heart. The aim of this project was to investigate the properties of cardiac conduction in a co-culture approach using SCMs and PCMs in cultured cell strands. Murine embryonic SCMs were pooled with fetal ventricular cells and seeded in predefined proportions on microelectrode arrays to form patterned strands of mixed cells. Conduction velocity (CV) was measured during steady state pacing. SCM excitability was estimated from action potentials measured in single cells using the patch clamp technique. Experiments were complemented with computer simulations of conduction using a detailed model of cellular architecture in mixed cell strands. CV was significantly lower in strands composed purely of SCMs (5.5 ± 1.5 cm/s, n = 11) as compared to PCMs (34.9 ± 2.9 cm/s, n = 21) at similar refractoriness (100% SCMs: 122 ± 25 ms, n = 9; 100% PCMs: 139 ± 67 ms, n = 14). In mixed strands combining both cell types, CV was higher than in pure SCMs strands, but always lower than in 100% PCM strands. Computer simulations demonstrated that both intercellular coupling and electrical excitability limit CV. These data provide evidence that in cultures of murine ventricular cardiomyocytes, SCMs cannot restore CV to control levels resulting in slow conduction, which may lead to reentry circuits and arrhythmias.

Functional Characterization and Comparison of Intercellular Communication in Stem Cell-Derived Cardiomyocytes

One novel treatment strategy for the diseased heart focuses on the use of pluripotent stem cell-derived cardiomyocytes (SC-CMs) to overcome the heart's innate deficiency for self-repair. However, targeted application of SC-CMs requires in-depth characterization of their true cardiogenic potential in terms of excitability and intercellular coupling at cellular level and in multicellular preparations. In this study, we elucidated the electrical characteristics of single SC-CMs and intercellular coupling quality of cell pairs, and concomitantly compared them with well-characterized murine native neonatal and immortalized HL-1 cardiomyocytes. Firstly, we investigated the electrical properties and Ca2+ signaling mechanisms specific to cardiac contraction in single SC-CMs. Despite heterogeneity of the new cardiac cell population, their electrophysiological activity and Ca2+ handling were similar to native cells. Secondly, we investigated the capability of paired SC-CMs to form an adequate subunit of a functional syncytium and analyzed gap junctions and signal transmission by dye transfer in cell pairs. We discovered significantly diminished coupling in SC-CMs compared with native cells, which could not be enhanced by a coculture approach combining SC-CMs and primary CMs. Moreover, quantitative and structural analysis of gap junctions presented significantly reduced connexin expression levels compared with native CMs. Strong dependence of intercellular coupling on gap junction density was further confirmed by computational simulations. These novel findings demonstrate that despite the cardiogenic electrophysiological profile, SC-CMs present significant limitations in intercellular communication. Inadequate coupling may severely impair functional integration and signal transmission, which needs to be carefully considered for the prospective use of SC-CMs in cardiac repair.

Stem cell mobilization chemotherapy with gemcitabine is effective and safe in myeloma patients with bortezomib-induced neurotoxicity.

Vinorelbine chemotherapy with granulocyte-colony stimulating factor (G-CSF) stimulation is a widely applied non-myelosuppressive mobilization regimen in Switzerland for myeloma patients, but its neurotoxic potential limits its use in patients with bortezomib-induced polyneuropathy. In this single-center study, we alternatively evaluated safety and effectiveness of gemcitabine chemotherapy with G-CSF for mobilization of autologous stem cells. Between March 2012 and February 2013, all bortezomib-pretreated myeloma patients planned to undergo first-line high-dose melphalan chemotherapy received a single dose of 1250 mg/m(2) gemcitabine, with G-CSF started on day 4. The 24 patients in this study had received a median of four cycles of bortezomib-dexamethason-based induction. Bortezomib-related polyneuropathy was identified in 21 patients (88%) by clinical evaluation and a standardized questionnaire. Administration of gemcitabine mobilization did not induce new or aggravate pre-existing neuropathy. Stem cell mobilization was successful in all 24 patients, with a single day of apheresis being sufficient in 19 patients (78%). The median yield was 9.51 × 10(6) CD34+ cells/kg. Stem collection could be accomplished at day 8 in 67%. Our data suggest that single-dose gemcitabine together with G-CSF is an effective mobilization regimen in myeloma patients and a safe alternative non-myelosuppressive mobilization chemotherapy for myeloma patients with bortezomib-induced polyneuropathy.

Neurotoxicity of stem cell mobilization chemotherapy with vinorelbine in myeloma patients after bortezomib treatment.

Vinorelbine chemotherapy with G-CSF stimulation is the standard mobilization regimen in Switzerland for multiple myeloma patients. However, with the increasing use of bortezomib during induction treatment, adding the neurotoxic compound vinorelbine for mobilization may aggravate bortezomib-induced polyneuropathy. In this retrospective single-center study, we aimed to explore vinorelbine mediated neuropathy in 106 consecutive bortezomib pretreated myeloma patients. We confirmed that vinorelbine with G-CSF represents a reliable and effective regimen for mobilization of autologous stem cells. However, the single administration of 35 mg/m(2) vinorelbine added significant neurotoxicity. We found that 24 patients (24%) reported vinorelbine mediated neurotoxicity: Aggravation of bortezomib-induced neuropathy was observed in 17 patients (17%), and vinorelbine mobilization induced first occurrence of polyneuropathy in additional 7 patients (7%). We observed that development of polyneuropathy was not associated with differing survival rates. Finally, affected patients reported polyneuropathy associated disease burden as "very high" in 13% and "high" in 50%. Our data indicate that a single administration of vinorelbine to mobilize autologous stem cells is associated with significant additional polyneuropathy in bortezomib pretreated myeloma patients. The efficacy of vinorelbine mobilization should be balanced against its neurotoxic potential.

Human lung-derived mesenchymal stem cell-conditioned medium exerts in vitro antitumor effects in malignant pleural mesothelioma cell lines.

BACKGROUND The soluble factors secreted by mesenchymal stem cells are thought to either support or inhibit tumor growth. Herein, we investigated whether the human lung-derived mesenchymal stem cell-conditioned medium (hlMSC-CM) exerts antitumor activity in malignant pleural mesothelioma cell lines H28, H2052 and Meso4. METHODS hlMSC-CM was collected from the human lung-derived mesenchymal stem cells. Inhibition of tumor cell growth was based on the reduction of cell viability and inhibition of cell proliferation using the XTT and BrdU assays, respectively. Elimination of tumor spheroids was assessed by the anchorage-independent sphere formation assay. The cytokine profile of hlMSC-CM was determined by a chemiluminescence-based cytokine array. RESULTS Our data showed that hlMSC-CM contains a broad range of soluble factors which include: cytokines, chemokines, hormones, growth and angiogenic factors, matrix metalloproteinases, metalloproteinase inhibitors and cell-cell mediator proteins. The 48- and 72-hour hlMSC-CM treatments of H28, H2052 and Meso4 cell lines elicited significant decreases in cell viability and inhibited cell proliferation. The 72-hour hlMSC-CM incubation of H28 cells completely eliminated the drug-resistant sphere-forming cells, which is more potent than twice the half maximal inhibitory concentration of cisplatin. CONCLUSIONS Our findings indicate that the cell-free hlMSC-CM confers in vitro antitumor activities via soluble factors in the tested mesothelioma cells and, hence, may serve as a therapeutic tool to augment the current treatment strategies in malignant pleural mesothelioma.

The European Hematology Association Roadmap for European Hematology Research: a consensus document

Abstract The European Hematology Association (EHA) Roadmap for European Hematology Research highlights major achievements in diagnosis and treatment of blood disorders and identifies the greatest unmet clinical and scientific needs in those areas to enable better funded, more focused European hematology research. Initiated by the EHA, around 300 experts contributed to the consensus document, which will help European policy makers, research funders, research organizations, researchers, and patient groups make better informed decisions on hematology research. It also aims to raise public awareness of the burden of blood disorders on European society, which purely in economic terms is estimated at Euro 23 billion per year, a level of cost that is not matched in current European hematology research funding. In recent decades, hematology research has improved our fundamental understanding of the biology of blood disorders, and has improved diagnostics and treatments, sometimes in revolutionary ways. This progress highlights the potential of focused basic research programs such as this EHA Roadmap. The EHA Roadmap identifies nine sections in hematology: normal hematopoiesis, malignant lymphoid and myeloid diseases, anemias and related diseases, platelet disorders, blood coagulation and hemostatic disorders, transfusion medicine, infections in hematology, and hematopoietic stem cell transplantation. These sections span 60 smaller groups of diseases or disorders. The EHA Roadmap identifies priorities and needs across the field of hematology, including those to develop targeted therapies based on genomic profiling and chemical biology, to eradicate minimal residual malignant disease, and to develop cellular immunotherapies, combination treatments, gene therapies, hematopoietic stem cell treatments, and treatments that are better tolerated by elderly patients. Received December 15, 2015. Accepted January 27, 2016. Copyright © 2016, Ferrata Storti Foundation

Spatial Distribution of Stem Cell-Like Keratinocytes in Dissected Compound Hair Follicles of the Dog

Hair cycle disturbances are common in dogs and comparable to some alopecic disorders in humans. A normal hair cycle is maintained by follicular stem cells which are predominately found in an area known as the bulge. Due to similar morphological characteristics of the bulge area in humans and dogs, the shared particularity of compound hair follicles as well as similarities in follicular biomarker expression, the dog is a promising model to study human hair cycle and stem cell disorders. To gain insight into the spatial distribution of follicular keratinocytes with stem cell potential in canine compound follicles, we microdissected hair follicles in anagen and telogen from skin samples of freshly euthanized dogs. The keratinocytes isolated from different locations were investigated for their colony forming efficiency, growth and differentiation potential as well as clonal growth. Our results indicate that i) compound and single hair follicles exhibit a comparable spatial distribution pattern with respect to cells with high growth potential and stem cell-like characteristics, ii) the lower isthmus (comprising the bulge) harbors most cells with high growth potential in both, the anagen and the telogen hair cycle stage, iii) unlike in other species, colonies with highest growth potential are rather small with an irregular perimeter and iv) the keratinocytes derived from the bulbar region exhibit characteristics of actively dividing transit amplifying cells. Our results now provide the basis to conduct comparative studies of normal dogs and those with hair cycle disorders with the possibility to extend relevant findings to human patients.

Differentiation of Human Embryonic Stem Cell (hESC) Derived Pyramidal Neurons

The mammalian cerebral neocortex is a complex six-layered structure containing multiple types of neurons. Pyramidal neurons of the neocortex are formed during development in an inside-out manner, by which deep layer (DL) neurons are generated first, and upper layer (UL) neurons are generated last. Neurons within the six-layered neocortex express unique markers for their position, showing whether they are subplate, deep layer, upper layer, or Cajal-Retzius neurons. The sequential generation of cortical layers, which exists in vivo, has been partially recapitulated in vitro by differentiation of mouse embryonic stem cells (Gaspard et al., 2008) and human embryonic stem cells (hESC) (Eiraku et al., 2008). The timeline of generation of cortical neurons from hESC is still not well defined, and could be very important in the future of cell therapy. In this study we will define timeline for UL and DL neurons for our experimental paradigm as well as test the effects of fibroblast growth factors (FGF) 2 and 8 on this neuronal differentiation. Recent papers suggest that FGFs are critical for forebrain patterning (Storm et al., 2003). Neuronal differentiation after treatment with either FGF2 or FGF8 from hESCs will be examined and the proportion of specific neuronal markers will be analyzed using immunocytochemistry. Our results show that the generated pyramidal neurons will express DL and UL laminar markers in vitro as they do in vivo and that the presence of FGF8 in induction media creates a proliferative effect, while FGF2 induces hESC to differentiate at a higher rate.