Tenascin-C and tenascin-W in whisker follicle stem cell niches: possible roles in regulating stem cell proliferation and migration

The whisker follicle has CD34-positive stem cells that migrate from their niche near the bulge along the glassy membrane to the whisker bulb, where they participate in the formation of the whisker shaft. Using immunohistochemistry we found the glycoprotein tenascin-C in the fibrous capsule of mouse whisker follicles, along the glassy membrane and in the trabecular region surrounding keratin-15-negative, CD34-positive stem cells. The related glycoprotein tenascin-W is found in the CD34-positive stem cell niche, in nearby trabeculae, and along the glassy membrane. Tenascin-W is also found in the neural stem cell niche of nearby hair follicles. The formation of stress fibers and focal adhesion complexes in CD34-positive whisker-derived stem cells cultured on fibronectin was inhibited by both tenascin-C and tenascin-W, which is consistent with a role for these glycoproteins in promoting the migration of these cells from the niche to the whisker bulb. Tenascin-C, but not tenascin-W, increased the proliferation of whisker follicle stem cells in vitro. Thus, the CD34-positive whisker follicle stem cell niche contains both tenascin-C and tenascin-W, and these glycoproteins may play a role in directing the migration and proliferation of these stem cells.

3D scaffolds co-seeded with human endothelial progenitor and mesenchymal stem cells: evidence of prevascularisation within 7 days

Blood supply is a critical issue in most tissue engineering approaches for large defect healing. As vessel ingrowth from surrounding tissues is proven to be insufficient, current strategies are focusing on the neo-vascularisation process. In the present study, we developed an in vitro pre-vascularised construct using 3D polyurethane (PU) scaffolds, based on the association of human Endothelial Progenitor Cells (EPC, CD34+ and CD133+) with human Mesenchymal Stem Cells (MSC). We showed the formation of luminal tubular structures in the co-seeded scaffolds as early as day 7 in culture. These tubular structures were proven positive for endothelial markers von Willebrand Factor and PECAM-1. Of special significance in our constructs is the presence of CD146-positive cells, as a part of the neovasculature scaffolding. These cells, coming from the mesenchymal stem cells population (MSC or EPC-depleted MSC), also expressed other markers of pericyte cells (NG2 and αSMA) that are known to play a pivotal function in the stabilisation of newly formed pre-vascular networks. In parallel, in co-cultures, osteogenic differentiation of MSCs occurred earlier when compared to MSCs monocultures, suggesting the close cooperation between the two cell populations. The presence of angiogenic factors (from autologous platelet lysates) in association with osteogenic factors seems to be crucial for both cell populations' cooperation. These results are promising for future clinical applications, as all components (cells, growth factors) can be prepared in an autologous way.

G-CSF induced arteriogenesis in humans: molecular insights into a randomized controlled trial

AIMS Recent data have demonstrated the feasibility of therapeutic induction of coronary collateral growth (arteriogenesis); however, mechanisms of action of such therapeutic collateral stimulation in humans are unknown. The aim of this study was to evaluate potential mechanisms, especially the involvement of arteriogenesis-relevant genes. METHODS AND RESULTS A total of 52 patients were randomized into two groups: subcutaneous G-CSF (10 μg/kg; n=26) or placebo (n=26). Before and after this 2-week treatment, collateral-flow index (CFI) was determined by simultaneous measurement of mean aortic, distal coronary occlusive and central venous pressure. CD34+ endothelial progenitor cells (EPC) and monocytes were quantified before, during and after treatment; gene-expression analysis of monocytes was performed with real-time polymerase chain reaction (RT-PCR). G-CSF lead to a significant increase of EPC and monocytes (4.8 and 2.6 fold, p < 0.05); for both cell types, the extent of increase correlated with CFI increase (r=0.23 and 0.14, p < 0.05). G-CSF also induced a change in gene expression of pro-and anti-arteriogenic genes in monocytes. Among nine assessed genes, three were found to be differentially regulated (IL8, JAK2, and PNPLa4; p < 0.05). CONCLUSIONS The mechanism of induction of collateral growth by G-CSF is related to an increase of EPC and of peripheral monocytes. It also leads to a change toward a pro-arteriogenic gene expression in peripheral monocytes.

Cytotoxic T cells induce proliferation of chronic myeloid leukemia stem cells by secreting interferon-γ

Chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasia arising from the oncogenic break point cluster region/Abelson murine leukemia viral oncogene homolog 1 translocation in hematopoietic stem cells (HSCs), resulting in a leukemia stem cell (LSC). Curing CML depends on the eradication of LSCs. Unfortunately, LSCs are resistant to current treatment strategies. The host’s immune system is thought to contribute to disease control, and several immunotherapy strategies are under investigation. However, the interaction of the immune system with LSCs is poorly defined. In the present study, we use a murine CML model to show that LSCs express major histocompatibility complex (MHC) and co-stimulatory molecules and are recognized and killed by leukemia-specific CD8+ effector CTLs in vitro. In contrast, therapeutic infusions of effector CTLs into CML mice in vivo failed to eradicate LSCs but, paradoxically, increased LSC numbers. LSC proliferation and differentiation was induced by CTL-secreted IFN-γ. Effector CTLs were only able to eliminate LSCs in a situation with minimal leukemia load where CTL-secreted IFN-γ levels were low. In addition, IFN-γ increased proliferation and colony formation of CD34+ stem/progenitor cells from CML patients in vitro. Our study reveals a novel mechanism by which the immune system contributes to leukemia progression and may be important to improve T cell–based immunotherapy against leukemia.

Collagenous Myofibroblastic Tumor of the Mandible: Case Report of a Unique Locally Aggressive Neoplasm Journal Head and Neck Pathology

We report a locally aggressive collagenous myofibroblastic neoplasm of the mandible in an 18-year-old male. Clinically, the lesion presented with rapid growth and irregular mandibular bone destruction. Grossly, the tumor was 10 cm in greatest dimension, light-tan, firm, and involving the posterior one-thirds of the body and inferior half of the left mandibular ramus. Histologically, the lesion was composed of a loose spindle cell proliferation interspersed with periodic dense bands of collagen. The spindle cells reacted positively to smooth muscle actin, calponin, and focally to desmin and were negative for S-100, pan-cytokeratin, CD99, CD34 and caldesmon, supporting myofibroblastic derivation. At our 4 year follow-up, the patient remained free of local recurrence and surgery related complications. The clinicopathologic findings and the differential diagnosis of this lesion is presented and discussed.

Gene transfer by viral vectors

To initiate our clinical trial for chemotherapy protection, I established the retroviral vector system for human MDR1 cDNA gene transfer. The human MDR1 cDNA continued to be expressed in the transduced bone marrow cells after four cohorts of serial transplants, 17 months after the initial transduction and transplant. In addition, we used this retroviral vector pVMDR1 to transduce human bone marrow and peripheral blood CD34$\sp+$ cells on stromal monolayer in the presence of hematopoietic growth factors. These data suggest that the retroviral vector pVMDR1 could modify hematopoietic precursor cells with a capacity for long-term self renewal. Thus, it may be possible to use the MDR1 retroviruses to confer chemotherapeutic protection on human normal hematopoietic precursor cells of ovarian and breast cancer patients in whom high doses of MDR drugs may be required to control the diseases.^ Another promising vector system is recombinant adeno-associated virus (rAAV) vector. An impediment to use rAAV vectors is that production of rAAV vectors for clinical use is extremely cumbersome and labor intensive. First I set up the rAAV vector system in our laboratory and then, I focused on studies related to the production of rAAV vectors for clinical use. By using a self-inactivating retroviral vector carrying a selection marker under the control of the CMV immediate early promoter and an AAV genome with the deletion of both ITRs, I have developed either a transient or a stable method to produce rAAV vectors. These methods involve infection only and can generate high-titer rAAV vectors (up to 2 x 10$\sp5$ cfu/ml of CVL) with much less work.^ Although recombinant adenoviral vectors hardly infect early hematopoietic precursor cells lacking $\alpha\sb v\beta\sb5$ or $\alpha\sb v\beta\sb3$ integrin on their surface, but efficiently infect other cells, we can use these properties of adenoviral vectors for bone marrow purging as well as for development of new viral vectors such as pseudotyped retroviral vectors and rAAV vectors. Replacement of self-inactivating retroviral vectors by recombinant adenoviral vectors will facilitate the above strategies for production of new viral vectors. In order to accomplish these goals, I developed a new method which is much more efficient than the current methods to construct adenoviral vectors. This method involves a cosmid vector system which is utilized to construct the full-length recombinant adenoviral vectors in vitro.^ First, I developed an efficient and flexible method for in vitro construction of the full-length recombinant adenoviral vectors in the cosmid vector system by use of a three-DNA fragment ligation. Then, this system was improved by use of a two-DNA fragment ligation. The cloning capacity of recombinant adenoviral vectors constructed by this method to develop recombinant adenoviral vectors depends on the efficiency of transfection only. No homologous recombination is required for development of infectious adenoviral vectors. Thus, the efficiency of generating the recombinant adenoviral vectors by the cosmid method reported here was much higher than that by the in vitro direct ligation method or the in vivo homologous recombination method reported before. This method of the in vitro construction of recombinant adenoviral vectors in the cosmid vector system may facilitate the development of adenoviral vector for human gene therapy. (Abstract shortened by UMI.) ^

Preeclampsia enhances neuroglial marker expression in umbilical cord Wharton's jelly-derived mesenchymal stem cells.

Objective: The aim of the study was to compare the neuroglial phenotype of Wharton's jelly-derived mesenchymal stem cells (WJ-MSC) from pregnancies complicated with preeclampsia and gestational age (GA)-matched controls. Methods: WJ-MSC were isolated from umbilical cords from both groups and analyzed for the cell surface expression of MSC markers and the gene and protein expression of neuroglial markers. Results: All WJ cells were highly positive for the MSC markers CD105, CD90 and CD73, but negative for markers specific for hematopoietic (CD34) and immunological cells (CD45, CD14, CD19 and HLA-DR). WJ-MSC from both groups expressed neuroglial markers (MAP-2, GFAP, MBP, Musashi-1 and Nestin) at the mRNA and protein level. The protein expressions of neuronal (MAP-2) and oligodendrocytic (MBP) markers were significantly increased in WJ-MSC from preeclampsia versus GA-matched controls. Conclusions: WJ-MSC from preeclamptic patients are possibly more committed to neuroglial differentiation through the activation of pathways involved both in the pathophysiology of the disease and in neurogenesis.

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.

Delayed Haematological recovery after autologous stem cell transplantation is associated with favourable outcome in acute myeloid leukaemia.

Autologous stem cell transplantation (ASCT) is applied to consolidate first remission in patients with acute myeloid leukaemia (AML). However, outcome after ASCT widely varies among AML patients. We analyzed the prognostic significance of haematological recovery for neutrophils [absolute neutrophil count (ANC) >1·0 × 10(9) /l] and platelets (platelet count >20·0 × 10(9) /l), stratifying at day 20 after ASCT in 88 consecutive and homogeneously treated AML patients in first remission. We observed that patients with delayed recovery had better overall survival (OS; ANC: P < 0·0001 and platelets: P = 0·0062) and time to progression (TTP; ANC: P = 0·0003 and platelets: P = 0·0125). Delayed recovery was an independent marker for better OS and TTP in a multivariate analysis including age, gender, number of transfused CD34+ cells, cytogenetics, FLT3-internal tandem duplication and NPM1 mutation. Our results suggest that delayed neutrophil and platelet recovery is associated with longer OS and TTP in AML patients consolidated with ASCT in first remission.

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.

Role of plerixafor in autologous stem cell mobilization with vinorelbine chemotherapy and granulocyte-colony stimulating factor in patients with myeloma: a phase II study (PAV-trial)

Current practice in Switzerland for the mobilization of autologous stem cells in patients with myeloma is combining vinorelbine chemotherapy and granulocyte-colony stimulating factor (G-CSF) cytokine stimulation. We prospectively investigated adding intravenous plerixafor to the vinorelbine/G-CSF combination (VGP), and compared it with vinorelbine/plerixafor (VP) and G-CSF/plerixafor (GP) combinations. In a final cohort (VP-late), plerixafor was given on the first day of CD34 + cells increasing to > 15 000/mL peripheral blood. Four consecutive cohorts of 10 patients with myeloma were studied. We observed that intravenously administered plerixafor can be safely combined with vinorelbine/G-CSF. VGP was superior in mobilizing peripheral stem and progenitor cells compared to the three double combinations (VP, GP and VP-late), and GP mobilized better than VP. Our data indicate that the triple combination of VGP is an efficient strategy to collect autologous CD34 + cells, with G-CSF contributing predominantly in this concept. Plerixafor can be safely added to G-CSF and/or vinorelbine chemotherapy.

Induction of the autophagy-associated gene MAP1S via PU.1 supports APL differentiation

The PU.1 transcription factor is essential for myeloid development. We investigated if the microtubule-associated protein 1S (MAP1S) is a novel PU.1 target with a link to autophagy, a cellular recycling pathway. Comparable to PU.1, MAP1S expression was significantly repressed in primary AML blasts as compared to mature neutrophils. Accordingly, MAP1S expression was induced during neutrophil differentiation of CD34(+) progenitor and APL cells. Moreover, PU.1 bound to the MAP1S promoter and induced MAP1S expression during APL differentiation. Inhibiting MAP1S resulted in aberrant neutrophil differentiation and autophagy. Taken together, our findings implicate the PU.1-regulated MAP1S gene in neutrophil differentiation and autophagy control.

p62/SQSTM1 upregulation constitutes a survival mechanism that occurs during granulocytic differentiation of acute myeloid leukemia cells.

The p62/SQSTM1 adapter protein has an important role in the regulation of several key signaling pathways and helps transport ubiquitinated proteins to the autophagosomes and proteasome for degradation. Here, we investigate the regulation and roles of p62/SQSTM1 during acute myeloid leukemia (AML) cell maturation into granulocytes. Levels of p62/SQSTM1 mRNA and protein were both significantly increased during all-trans retinoic acid (ATRA)-induced differentiation of AML cells through a mechanism that depends on NF-κB activation. We show that this response constitutes a survival mechanism that prolongs the life span of mature AML cells and mitigates the effects of accumulation of aggregated proteins that occurs during granulocytic differentiation. Interestingly, ATRA-induced p62/SQSTM1 upregulation was impaired in maturation-resistant AML cells but was reactivated when differentiation was restored in these cells. Primary blast cells of AML patients and CD34(+) progenitors exhibited significantly lower p62/SQSTM1 mRNA levels than did mature granulocytes from healthy donors. Our results demonstrate that p62/SQSTM1 expression is upregulated in mature compared with immature myeloid cells and reveal a pro-survival function of the NF-κB/SQSTM1 signaling axis during granulocytic differentiation of AML cells. These findings may help our understanding of neutrophil/granulocyte development and will guide the development of novel therapeutic strategies for refractory and relapsed AML patients with previous exposure to ATRA.

Retinal differentiation of human bone marrow-derived stem cells by co-culture with retinal pigment epithelium in vitro.

The goal of this study was to assess the in vitro differentiation capacity of human bone marrow-derived stem cells (hBMSCs) along retinal lineages. Mononuclear cells (MNC) were isolated from bone marrow (BM) and mobilized peripheral blood (mPB) using Ficoll-Paque density gradient centrifugation, and were sorted by magnetic-activated cell sorting (MACS) for specific stem cell subsets (CD34(+)CD38(+)/CD34(+)CD38(-)). These cells were then co-cultured on human retinal pigment epithelial cells (hRPE) for 7 days. The expression of stem cell, neural and retina-specific markers was examined by immunostaining, and the gene expression profiles were assessed after FACS separation of the co-cultured hBMSCs by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, in vitro functionality of the differentiated cells was analyzed by quantifying phagocytosis of CY5-labeled photoreceptor outer segments (POS). After 7 days of co-culture, hBMSCs adopted an elongated epithelial-like morphology and expressed RPE-specific markers, such as RPE65 and bestrophin. In addition, these differentiated cells were able to phagocytose OS, one of the main characteristics of native RPE cells. Our data demonstrated that human CD34(+)CD38(-) hBMSC may differentiate towards an RPE-like cell type in vitro and could become a new type of autologous donor cell for regenerative therapy in retinal degenerative diseases.

"Leiomyomatoid angiomatous neuroendocrine tumor" (LANT) of the pituitary reflects idiosyncratic angiogenesis in adenomas of the gonadotroph cell lineage.

Based on a single-case observation, the descriptive label "leiomyomatoid angiomatous neuroendocrine tumor" (LANT) has been tentatively applied to what was perceived as a possible novel type of dual-lineage pituitary neoplasm with biphasic architecture. We report on two additional examples of an analogous phenomenon encountered in male patients, aged 59 years (Case 1) and 91 years (Case 2). Both tumors were intra- and suprasellar masses, measuring 5.6 cm × 4.4 cm × 3.4 cm, and 2.7 cm × 2 cm × 1.7 cm, respectively. Histologically, Case 1 was an FSH-cell adenoma interwoven by vascularized connective tissue septa that tended to exhibit incremental stages of adventitial overgrowth. The epithelial component of Case 2 corresponded to an LH-cell adenoma, and lay partitioned by a maze of paucicellular to hyalinized vascular axes. Irrespective of architectural variations, perivascular spindle cells exhibited immunopositivity for vimentin, muscular actin, and smooth muscle actin. Conversely, negative results were obtained for CD34, EMA, S100 protein, GFAP, and TTF-1. Ultrastructural study failed to reveal metaplastic cell forms involving transitional features between adenohypophyseal-epithelial and mesenchymal-contractile phenotype. We propose that LANT be regarded as a peculiar reflection of maladaptive angiogenesis in some pituitary adenomas, rather than a genuine hybrid neoplasm. While no mechanistic clue is forthcoming to account for this distinctive pattern, hemodynamic strain through direct arterial - rather than portal - supply of the adenoma's capillary bed may be one such explanatory factor. The apparent predilection of the LANT pattern for macroadenomas of the gonadotroph cell lineage remains unexplained.