An in vivo comparative study of sonic, desert and Indian hedgehog reveals that hedgehog pathway activity regulates epidermal stem cell homeostasis

Despite the well-characterised role of sonic hedgehog (Shh) in promoting interfollicular basal cell proliferation and hair follicle downgrowth, the role of hedgehog signalling during epidermal stem cell fate remains largely uncharacterised. In order to determine whether the three vertebrate hedgehog molecules play a role in regulating epidermal renewal we overexpressed sonic (Shh), desert (Dhh) and Indian (Ihh) hedgehog in the basal cells of mouse skin under the control of the human keratin 14 promoter. We observed no overt epidermal morphogenesis phenotype in response to Ihh overexpression, however Dhh overexpression resulted in a range of embryonic and adult skin manifestations indistinguishable from Shh overexpression. Two distinct novel phenotypes were observed amongst Shh and Dhh transgenics, one exhibiting epidermal progenitor cell hyperplasia with the other displaying a complete loss of epidermal tissue renewal indicating deregulation of stem cell activity. These data suggest that correct temporal regulation of hedgehog activity is a key factor in ensuring epidermal stem cell maintenance. In addition, we observed Shh and Dhh transgenic skin from both phenotypes developed lesions reminiscent of human basal cell carcinoma (BCC), indicating that BCCs can be generated despite the loss of much of the proliferative (basal) compartment. These data suggest the intriguing possibility that BCC can arise outside the stem cell population. Thus the elucidation of Shh (and Dhh) target gene activation in the skin will likely identify those genes responsible for increasing the proliferative potential of epidermal basal cells and the mechanisms involved in regulating epidermal stem cell fate.

Autologous hemopoietic stem cell transplantation in severe rheumatoid arthritis: A report from the EBMT and ABMTR

Objective. Since 1996, autologous hemopoietic stem cell transplantation (HSCT) has been used to treat severe rheumatoid arthritis (RA). To date, published reports have been individual cases or series containing small numbers. This study combined the worldwide experience in a single analysis. Methods. The Autoimmune Disease Databases of the European Group for Blood and Marrow Transplantation (EBMT) and the Autologous Blood and Marrow Transplant Registry (ABMTR) were used to identify patients with RA treated with autologous HSCT. Further information relating to patient and treatment-specific variables was obtained by questionnaire. Results. Seventy-six patients were registered from 15 centers. Seventy-three patients had received autologous HSCT, and in 3 patients hematopoietic stem cells (HSC) were mobilized but not transplanted. Transplanted patients (median age 42 yrs, 74% female, 86% rheumatoid factor positive) had been previously treated with a mean of 5 (range 2-9) disease modifying antirheumatic drugs (DMARD). Significant functional impairment was present, with a median Health Assessment Questionnaire (HAQ) score of 1.4 (range 1.1-2.0) and Steinbrocker score mean 2.39 (SD 0.58). The high dose treatment regimen was cyclophosphamide (CYC) alone in the majority of patients, mostly 200 mg/kg (n = 62). Seven patients received anti-thymocyte globulin (ATG) in addition to CYC, 2 patients busulfan and CYC (BuCYC), and one patient CYC with total body irradiation and ATG. One patient received fludarabine with ATG. Following treatment, one patient received bone marrow but the rest received chemotherapy and/or granulocyte colony-stimulating factor mobilized peripheral blood stem cells. The harvest was unmanipulated in 28 patients, the rest receiving some form of lymphocyte depletion, mostly through CD34+ selection. Median followup was 16 months (range 3-55). Responses were measured using the American College of Rheumatology (ACR) criteria. Forty-nine patients (67%) achieved at least ACR 50% response at some point following transplant. There was a significant reduction in the level of disability measured by the HAQ (p < 0.005). Most patients restarted DMARD within 6 months for persistent or recurrent disease activity, which provided disease control in about half the cases. Response was significantly related to seronegative RA (p = 0.02) but not to duration of disease, number of previous DMARD, presence of HLA-DR4, or removal of lymphocytes from the graft. There was no direct transplant related mortality, although one patient, treated with the BuCYC regimen, died 5 months post-transplant from infection and incidental non-small cell lung cancer. Conclusion. Autologous HSCT is a relatively safe form of salvage treatment in severe, resistant RA. In these open label studies significant responses were achieved in most patients, with over 50% achieving an ACR 50 or more response at 12 months. Although the procedure is not curative, recurrent or persistent disease activity may be subsequently controlled in some patients with DMARD. Clinical trials are necessary to develop this approach inpatients with aggressive disease who have failed conventional treatment including anti-tumor necrosis factor agents.

The adult mouse hippocampal progenitor is neurogenic but not a stem cell

The aim of this investigation was to characterize the proliferative precursor cells in the adult mouse hippocampal region. Given that a very large number of new hippocampal cells are generated over the lifetime of an animal, it is predicted that a neural stem cell is ultimately responsible for maintaining this genesis. Although it is generally accepted that a proliferative precursor resides within the hippocampus, contradictory reports exist regarding the classification of this cell. Is it a true stem cell or a more limited progenitor? Using a strict functional definition of a neural stem cell and a number of in vitro assays, we report that the resident hippocampal precursor is a progenitor capable of proliferation and multipotential differentiation but is unable to self-renew and thus proliferate indefinitely. Furthermore, the mitogen FGF-2 stimulates proliferation of these cells to a greater extent than epidermal growth factor ( EGF). In addition, we found that BDNF was essential for the production of neurons from the hippocampal progenitor cells, being required during proliferation to trigger neuronal fate. In contrast, a bona fide neural stem cell was identified in the lateral wall of the lateral ventricle surrounding the hippocampus. Interestingly, EGF proved to be the stronger mitogenic factor for this cell, which was clearly a different precursor from the resident hippocampal progenitor. These results suggest that the stem cell ultimately responsible for adult hippocampal neurogenesis resides outside the hippocampus, producing progenitor cells that migrate into the neurogenic zones and proliferate to produce new neurons and glia.

Mechanisms of hematopoietic stem cell mobilization: When innate immunity assails the cells that make blood and bone

Mobilization is now used worldwide to collect large numbers of hematopoietic stem and progenitor cells (HSPCs) for transplantation. Although the first mobilizing agents were discovered largely by accident, discovery of more efficient mobilizing agents will require a better understanding of the molecular mechanisms responsible. During the past 5 years, a number of mechanisms have been identified, shedding new light on the dynamics of the hematopoietic system in vivo and on the intricate relationship between hematopoiesis, innate immunity, and bone. After briefly reviewing the mechanisms by which circulating HSPCs home into the bone marrow and what keeps them there, the current knowledge of mechanisms responsible for HSPC mobilization in response to hematopoietic growth factors such as granulocyte colony-stimulating factor, chemotherapy, chemokines, and polyanions will be discussed together with current strategies developed to further increase HSPC mobilization. (c) 2006 International Society for Experimental Hematology.

Scaffold-free, Human Mesenchymal Stem Cell-Based Tissue Engineered Blood Vessels.

Tissue-engineered blood vessels (TEBV) can serve as vascular grafts and may also play an important role in the development of organs-on-a-chip. Most TEBV construction involves scaffolding with biomaterials such as collagen gel or electrospun fibrous mesh. Hypothesizing that a scaffold-free TEBV may be advantageous, we constructed a tubular structure (1 mm i.d.) from aligned human mesenchymal cell sheets (hMSC) as the wall and human endothelial progenitor cell (hEPC) coating as the lumen. The burst pressure of the scaffold-free TEBV was above 200 mmHg after three weeks of sequential culture in a rotating wall bioreactor and perfusion at 6.8 dynes/cm(2). The interwoven organization of the cell layers and extensive extracellular matrix (ECM) formation of the hMSC-based TEBV resembled that of native blood vessels. The TEBV exhibited flow-mediated vasodilation, vasoconstriction after exposure to 1 μM phenylephrine and released nitric oxide in a manner similar to that of porcine femoral vein. HL-60 cells attached to the TEBV lumen after TNF-α activation to suggest a functional endothelium. This study demonstrates the potential of a hEPC endothelialized hMSC-based TEBV for drug screening.

Characterization of ALDH1A1 as a novel tumorigenic target mediating TAZ-induced lung tumorigenesis and stem cell phenotypes

Recent studies suggest that lung cancer stem cells (CSCs) may play major roles in lung cancer development, metastasis and drug resistance. Therefore, identification of lung CSC drivers may provide promising targets for lung cancer. TAZ (transcriptional co-activator with PDZ-binding motif) is a transcriptional co-activator and key downstream effector of the Hippo pathway, which plays critical roles in various biological processes. TAZ has been shown to be overexpressed in non-small cell lung cancer (NSCLC) and involved in tumorigenicity of lung epithelial cells. However, whether TAZ is a driver for lung CSCs and tumor formation in vivo is unknown. In addition, the molecular mechanism underlying TAZ-induced lung tumorigenesis remains to be determined. In this study, we provided evidence that constitutively active TAZ (TAZ-S89A) is a driver for lung tumorigenesis in vivo in mice and formation of lung CSC. Oncogenes upregulated in TAZ-overexpressing cells were identified with further validation. The most dramatically activated gene, Aldh1a1 (Aldehyde dehydrogenase 1 family member a1), a well-established CSC marker, showed that TAZ induces Aldh1a1 transcription by activating its promoter activity through interaction with the transcription factor TEA domain (TEAD) family member. Most significantly, inhibition of ALDH1A1 with its inhibitor A37 or CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) gene knockout in lung cancer cells suppressed lung tumorigenic and CSC phenotypes in vitro, and tumor formation in mice in vivo. In conclusion, this study identified TAZ as a novel inducer of lung CSCs and the first transcriptional activator of the stem cell marker ALDH1A1. Most significantly, we identified ALDH1A1 as a critical meditator of TAZ-induced tumorigenic and CSC phenotypes in lung cancer. Our studies provided preclinical data for targeting of TAZ-TEAD-ALDH1A1 signaling to inhibit CSC-induced lung tumorigenesis and drug resistance in the future.

Gene scanning of VDJH-amplified segments is a clinically relevant technique to detect contaminating tumor cells in the apheresis products of multiple myeloma patients undergoing autologous peripheral blood stem cell transplantation.

Contaminating tumour cells in apheresis products have proved to influence the outcome of patients with multiple myeloma (MM) undergoing autologous stem cell transplantation (APBSCT). The gene scanning of clonally rearranged VDJ segments of the heavy chain immunoglobulin gene (VDJH) is a reproducible and easy to perform technique that can be optimised for clinical laboratories. We used it to analyse the aphereses of 27 MM patients undergoing APBSCT with clonally detectable VDJH segments, and 14 of them yielded monoclonal peaks in at least one apheresis product. The presence of positive results was not related to any pre-transplant characteristics, except the age at diagnosis (lower in patients with negative products, P = 0.04). Moreover, a better pre-transplant response trended to associate with a negative result (P = 0.069). Patients with clonally free products were more likely to obtain a better response to transplant (complete remission, 54% vs 28%; >90% reduction in the M-component, 93% vs 43% P = 0.028). In addition, patients transplanted with polyclonal products had longer progression-free survival, (39 vs 19 months, P = 0.037) and overall survival (81% vs 28% at 5 years, P = 0.045) than those transplanted with monoclonal apheresis. In summary, the gene scanning of apheresis products is a useful and clinically relevant technique in MM transplanted patients.

Role for cell-to-cell communication in stem cell specification toward pancreatic progenitors: relevance to the design of novel therapies for diabetes.

Thesis (Ph.D.)--University of Washington, 2016-08

Adipose-derived stem cell adhesion on laminin-coated microcarriers improves commitment toward the cardiomyogenic lineage

International audience

Decreased Expression Of Stem Cell Markers By Simvastatin In 7,12-dimethylbenz(a)anthracene (dmba)-induced Breast Cancer.

Simvastatin, a competitive inhibitor of HMG-CoA reductase widely used in the treatment and prevention of hyperlipidemia-related diseases, has recently been associated to in vitro anticancer stem cell (CSC) actions. However, these effects have not been confirmed in vivo. To assess in vivo anti-CSC effects of simvastatin, female Sprague-Dawley rats with 7,12-dimethyl-benz(a)anthracene (DMBA)-induced mammary cancer and control animals were treated for 14 days with either simvastatin (20 or 40 mg/kg/day) or soybean oil (N = 60). Tumors and normal breast tissues were removed for pathologic examination and immunodetection of CSC markers. At 40 mg/kg/day, simvastatin significantly reduced tumor growth and the expression of most CSC markers. The reduction in tumor growth (80%) could not be explained solely by the decrease in CSCs, since the latter accounted for less than 10% of the neoplasia (differentiated cancer cells were also affected). Stem cells in normal, nonneoplastic breast tissues were not affected by simvastatin. Simvastatin was also associated with a significant decrease in proliferative activity but no increase in cell death. In conclusion, this is the first study to confirm simvastatin anti-CSC actions in vivo, further demonstrating that this effect is specific for neoplastic cells, but not restricted to CSCs, and most likely due to inhibition of cell proliferation.

Unusual Erythrocyte Split Chimerism In Pregnancy After Allogeneic Stem Cell Transplantation.

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Protocols for obtainment and isolation of two mesenchymal stem cell sources in sheep

PURPOSE: To evaluate different protocols to isolate stem cells from ovine umbilical cord blood and adipose tissue. METHODS: There were used 5 samples of umbilical blood and 5 samples of perirenal adipose tissue from 10 female sheep. All the samples were obtained through surgery, to harvest aseptic samples. There were used 3 protocols for obtainment and culture of umbilical cord blood stem cells and 4 protocols for ovine adipose tissue stem cells. RESULTS: It was possible to observe only one successful protocol for the obtainment of umbilical cord blood stem cells. When analyzing the techniques used to obtain adipose tissue stem cells, only one of the methods was effective as well. Through colony forming unit assay, there were obtained 58 colonies of cells after seven days in culture. Flow citometry tests revealed the cells were positive to CD44 and exhibited negative reaction to CD38, CD45, CD41/61. These cells showed a growth curve with very well defined phases LOG, LAG and PLATEAU. This phases are typically seem in mesenchymal stem cells growth curves. CONCLUSIONS: The isolation and culture of mesenchymal stem cells from ovine umbilical cord blood are complex and request more detailed assays. Stem cells from fat tissue sheep showed mesenchymal characteristics, according to their cell growth curve, ability to origin colonies of fibroblastoid cells and positive reactivity with the antibody CD44 by flow citometry.

Activation of Neural and Pluripotent Stem Cell Signatures Correlates with Increased Malignancy in Human Glioma

The presence of stem cell characteristics in glioma cells raises the possibility that mechanisms promoting the maintenance and self-renewal of tissue specific stem cells have a similar function in tumor cells. Here we characterized human gliomas of various malignancy grades for the expression of stem cell regulatory proteins. We show that cells in high grade glioma co-express an array of markers defining neural stem cells (NSCs) and that these proteins can fulfill similar functions in tumor cells as in NSCs. However, in contrast to NSCs glioma cells co-express neural proteins together with pluripotent stem cell markers, including the transcription factors Oct4, Sox2, Nanog and Klf4. In line with this finding, in high grade gliomas mesodermal-and endodermal-specific transcription factors were detected together with neural proteins, a combination of lineage markers not normally present in the central nervous system. Persistent presence of pluripotent stem cell traits could only be detected in solid tumors, and observations based on in vitro studies and xenograft transplantations in mice imply that this presence is dependent on the combined activity of intrinsic and extrinsic regulatory cues. Together these results demonstrate a general deregulated expression of neural and pluripotent stem cell traits in malignant human gliomas, and indicate that stem cell regulatory factors may provide significant targets for therapeutic strategies.

Does mobilization for autologous stem cell transplantation damage stromal layer formation?

Autologous hematopoietic stem cell transplantation (HSCT) has proved efficient to treat hematological malignancies. However, some patients fail to mobilize HSCs. It is known that the microenvironment may undergo damage after allogeneic HSCT. However little is known about how chemotherapy and growth factors contribute to this damage. We studied the stromal layer formation(SLF) and velocity before and after HSC mobilization, through long-term bone marrow culture from 22 patients and 10 healthy donors. Patients` SLF was similar at pre- (12/22)and post-mobilization (9/20), however for controls this occurred more at pre- mobilization (9/10; p=0.03). SLF velocity was higher at pre than post-mobilization in both groups. Leukemias and multiple myeloma showed faster growth of SLF than lymphomas at post-mobilization, the latter being similar to controls. These findings could be explained by less uncommitted HSC in controls than patients at post-mobilization. Control HSCs may migrate more in response to mobilization, resulting in a reduced population by those cells.

Constitutively Active Mutant D816VKit Induces Megakayocyte and Mast Cell Differentiation of Early Haemopoietic Cells from Murine Foetal Liver

Mutations of Kit at position D816 have been implicated in mastocytosis, acute myeloid leukaemia and germ cell tumours. Expression of this mutant Kit in cell lines results in factor-independent growth, differentiation and increased survival in vitro and tumourigenicity in vivo. Mutant D816VKit and wild-type Kit were expressed in murine primary haemopoietic cells and grown in stem cell factor (SCF) or the absence of factors. Expression of D816VKit did not lead to transformation as assessed by a colony assay, but resulted in enhanced differentiation of cells when compared to control cells. D816VKit induced an increase in the number of cells differentiating along the megakaryocyte lineage in the absence of factors. SCF had an added effect with an increase in differentiation of mast cells. Expression of wild-type Kit in the presence of SCF also failed to cause transformation and induced differentiation of mast cells and megakaryocytes. We conclude that constitutive expression of D816VKit in primary haemopoietic cells is not a sufficient transforming stimulus but leads to the survival and maturation of cells whose phenotype is influenced by the presence of SCF. (C) 2003 Elsevier Science Ltd. All rights reserved.