Comparative proteomics of colon cancer stem cells and differentiated tumor cells identifies BIRC6 as a potential therapeutic target

Patients with liver metastases from colon carcinoma show highly variable responses to chemotherapy and tumor recurrence is frequently observed. Therapy-resistant cancer stem cells have been implicated in drug resistance and tumor recurrence. However, the factors determining therapy resistance and tumor recurrence are poorly understood. The aim of this study was to gain insight into these mechanisms by comparing the proteomes of patient-derived cancer stem cell cultures and their differentiated isogenic offspring. We established colonosphere cultures derived from resection specimens of liver metastases in patients with colon cancer. These colonospheres, enriched for colon cancer stem cells, were used to establish isogenic cultures of stably differentiated nontumorigenic progeny. Proteomics based on one-dimensional gel electrophoresis coupled to nano liquid chromatography tandem MS was used to identify proteome differences between three of these paired cultures. The resulting data were analyzed using Ingenuity Pathway Software. Out of a total data set of 3048 identified proteins, 32 proteins were at least twofold up-regulated in the colon cancer stem cells when compared with the differentiated cells. Pathway analysis showed that "cell death " regulation is strikingly different between the two cell types. Interestingly, one of the top-up-regulated proteins was BIRC6, which belongs to the class of Inhibitor of Apoptosis Proteins. Knockdown of BIRC6 sensitized colon cancer stem cells against the chemotherapeutic drugs oxaliplatin and cisplatin. This study reveals that differentiation of colon cancer stem cells is accompanied by altered regulation of cell death pathways. We identified BIRC6 as an important mediator of cancer stem cell resistance against cisplatin and oxaliplatin. Targeting BIRC6, or other Inhibitors of Apoptosis Proteins, may help eradicating colon cancer stem cells.     

Adipose-derived stem cells: Pathophysiologic implications vs therapeutic potential in systemic sclerosis

Adipose-derived stem cells(ADSCs)residing in the stromal vascular fraction(SVF)of white adipose tissue are recently emerging as an alternative tool for stem cell-based therapy in systemic sclerosis(SSc),a complex connective tissue disorder affecting the skin and internal organs with fibrotic and vascular lesions.Several preclinical and clinical studies have reported promising therapeutic effects of fat grafting and autologous SVF/ADSC-based local treatment for facial and hand cutaneous manifestations of SSc patients.However,currently available data indicate that ADSCs may represent a double-edged sword in SSc,as they may exhibit a pro-fibrotic and anti-adipogenic phenotype,possibly behaving as an additional pathogenic source of pro-fibrotic myofibroblasts through the adipocyte-to-myofibroblast transition process.Thus,in the perspective of a larger employ of SSc-ADSCs for further therapeutic applications,it is important to definitely unravel whether these cells present a comparable phenotype and similar immunosuppressive,anti-inflammatory,anti-fibrotic and pro-angiogenic properties in respect to healthy ADSCs.In light of the dual role that ADSCs seem to play in SSc,this review will provide a summary of the most recent insights into the preclinical and clinical studies employing SVF and ADSCs for the treatment of the disease and,at the same time,will focus on the main findings highlighting the possible involvement of these stem cells in SSc-related fibrosis pathogenesis.     

Combination effects of mesenchymal stem cells transplantation and anodal transcranial direct current stimulation on a cuprizone-induced mouse model of multiple sclerosis

Journal of Molecular Histology - Multiple sclerosis (MS) has no absolute treatment, and researchers are still exploring to introduce promising therapy for MS. Transcranial direct current...     

Using stem cells in multiple sclerosis therapies

Stem cell transplantation approaches offer for the first time the opportunity to design therapeutic approaches for multiple sclerosis (MS) with curative intent. Here we discuss key observations and questions emerging from clinical trials of hematopoietic stem cell transplantation for MS and from studies of myelin/neural repair in experimental models of demyelinating disorders.     

Amniotic epithelial cells from the human placenta potently suppress a mouse model of multiple sclerosis

Human amniotic epithelial cells (hAEC) have stem cell-like features and immunomodulatory properties. Here we show that hAEC significantly suppressed splenocyte proliferation in vitro and potently attenuated a mouse model of multiple sclerosis (MS). Central nervous system (CNS) CD3(+) T cell and F4/80(+) monocyte/macrophage infiltration and demyelination were significantly reduced with hAEC treatment. Besides the known secretion of prostaglandin E2 (PGE2), we report the novel finding that hAEC utilize transforming growth factor-β (TGF-β) for immunosuppression. Neutralization of TGF-β or PGE2 in splenocyte proliferation assays significantly reduced hAEC-induced suppression. Splenocytes from hAEC-treated mice showed a Th2 cytokine shift with significantly elevated IL-5 production. While transferred CFSE-labeled hAEC could be detected in the lung, none were identified in the CNS or in lymphoid organs. This is the first report documenting the therapeutic effect of hAEC in a MS-like model and suggest that hAEC may have potential for use as therapy for MS.     

The therapeutic potential of stem cells

In recent years, there has been an explosion of interest in stem cells, not just within the scientific and medical communities but also among politicians, religious groups and ethicists. Here, we summarize the different types of stem cells that have been described: their origins in embryonic and adult tissues and their differentiation potential in vivo and in culture. We review some current clinical applications of stem cells, highlighting the problems encountered when going from proof-of-principle in the laboratory to widespread clinical practice. While some of the key genetic and epigenetic factors that determine stem cell properties have been identified, there is still much to be learned about how these factors interact. There is a growing realization of the importance of environmental factors in regulating stem cell behaviour and this is being explored by imaging stem cells in vivo and recreating artificial niches in vitro. New therapies, based on stem cell transplantation or endogenous stem cells, are emerging areas, as is drug discovery based on patient-specific pluripotent cells and cancer stem cells. What makes stem cell research so exciting is its tremendous potential to benefit human health and the opportunities for interdisciplinary research that it presents.     

The therapeutic potential of HDAC inhibitors in the treatment of multiple sclerosis

Multiple sclerosis (MS) is a devastating autoimmune disorder of the central nervous system (CNS) for which there is no efficacious cure. Thanks to numerous preclinical and clinical studies, drugs able to mitigate the inexorable course of the disease have been made available recently. Still, there is a terrible need for compounds capable of reducing the severity of the autoimmune attack and of blocking progression of the disorder. Also, besides the classic immunosuppressive strategies, it is now appreciated that compounds directly targeting neuronal death can be of relevance to the treatment of MS patients. Acetylation homeostasis is a key regulator of both immune cell activation and neuronal survival. Of note, potent histone deacetylase inhibitors (HDACi) endowed with antiinflammatory and neuroprotective properties have been identified. Efficacy of HDACi in experimental models of MS has been reported consistently. In this review, we provide an appraisal of the literature on HDACi and MS, also discussing the mechanisms by which HDACi can suppress the autoimmune attack to the CNS.     

Harnessing the therapeutic potential of myogenic stem cells

The potential clinical use of stem cells for cell transplantation therapies to replace defective genes in myopathies is an area of intense investigation. Precursor cells derived from non-muscle tissue with myogenic potential have been identified in many tissues, including bone marrow and dermis, although the status of these putative stem cells requires clarification. The incorporation of circulating bone-marrow derived stem cells into regenerating adult skeletal muscle has been demonstrated in mice but the contribution of donor cells is so minimal that it would appear clinically irrelevant at this stage. The possibility of a true stem cell subpopulation within skeletal muscle that replenishes the satellite cells (conventional muscle precursors on the surface of myofibres) is also very attractive as a superior source of myoblasts for muscle construction. A full understanding of the intrinsic factors (i.e. gene expression within the stem cell) and extrinsic factors (i.e. signals from the external environment) which control the commitment of stem cells to the myogenic lineage, and the conditions which favour stem cell expansion in vivo is required before stem cells can be seriously considered for clinical cell therapy. This revised version was published online in July 2006 with corrections to the Cover Date.     

Presynaptic dopaminergic properties of differentiated mouse embryonic stem cells

This study characterized the presynaptic dopaminergic properties of neuronally differentiated mouse embryonic stem (ES) cells. Approximately 30% of the ES cells expressed tyrosine hydroxylase (TH) immunoreactivity when co-cultured with PA6 cells. These cultures expressed high affinity, sodium-dependent dopamine uptake as well as depolarization-induced and calcium-dependent dopamine release of this transmitter. These and other important dopaminergic genes found expressed in these cultures by RT-PCR included Nurr1, vesicular monoamine transporter 2 (VMAT2), TH, dopamine transporter (DAT), and glial cell line-derived neurotrophic factor (GDNF) receptors c-Ret and GFRalpha1. These results demonstrate that differentiated ES cells have the presynaptic functions for maintaining dopaminergic homeostasis, which may be essential for their long-term use in restoring CNS levels of this transmitter.     

Neural precursor cells differentiated from mouse embryonic stem cells relieve symptomatic motor behavior in a rat model of Parkinson's disease

Pluripotent embryonic stem (ES) cells are the most versatile cells, with the potential to differentiate into all types of cell lineages including neural precursor cells (NPCs), which can be expanded in large numbers for significant periods of time to provide a reliable cell source for transplantation in neurodegenerative disorders such as Parkinson's disease (PD). In the present study, we used the MESPU35 mouse ES cell line, which expresses enhanced green fluorescent protein that enables one to distinguish between transplanted cells and cells of host origin. Embryoid bodies (EBs) were formed and were induced to NPCs in N2 selection medium plus fibronectin. Praxiology and immunohistochemistry methods were used to observe the survival, differentiation, and therapeutic effect of NPCs after grafted into the striatum of PD rats. We found that mouse ESc were differentiated into nestin-positive NPCs 6 days after the EBs formed and cultured in the N2 selection medium. The number of survival NPCs was increased significantly by fibronectin. About 23.76+/-2.29% of remaining cells were tyrosine hydroxylase (TH)-positive 12 days after NPCs were cultured in N2 selective medium. The survival rates of NPCs were 2.10+/-0.41% and about 90.90+/-3.00% of the engrafted NPCs were TH-positive 6 weeks after transplantation into the striatum of PD rats. The rotation of PD rats was relieved 3 weeks after the NPCs transplantation and this effect was kept for at least 6 weeks. It suggests that most of the survival NPCs derived from ES cells differentiated into TH-positive neurons after grafted into the striatum of PD rats, which produces therapeutic effect on PD.     

The therapeutic potential of neural stem cells

Recent evidence shows that transplantation of neural stem/precursor cells may protect the central nervous system from inflammatory damage through a 'bystander' mechanism that is alternative to cell replacement. This novel mechanism, which might improve the success of transplantation procedures, is exerted by undifferentiated neural stem cells, the functional characteristics of which are regulated by important stem cell regulators released by CNS-resident and blood-borne inflammatory cells. Here, we discuss this alternative bystander mechanism in the context of the atypical ectopic perivascular niche. We propose that it is the most challenging example of reciprocal therapeutic crosstalk between the inflamed CNS and systemically transplanted neural stem cells.     

小鼠胚胎干细胞分化为血管内皮细胞的永生化研究

本文探讨了小鼠胚胎干细胞（ES细胞）、诱导分化的血管内皮细胞永生化。在体外培养系统中,以维甲酸（RA）和转化生长因子－β1（TGF－β1）诱导小鼠胚胎干细胞（ES细胞）的拟胚体（EB）分化为“圆形细胞”和由这些“圆形细胞”组成的血管样结构。经光学和扫描电镜及免疫荧光等法分析检测,证明组成血管样结构的细胞具有专一性vWF荧光染色,表明是血管内皮样细胞。利用脂质体将人端粒酶催化亚基逆转录酶（hTERT)基因转染诱导分化中的“圆形细胞”。应用Dot-blot,RT－PCR,Western blot及免疫组织化学等方法分析、观察和证明了诱导分化的组成血管样结构的园形细胞和被hTERT基因转染的“圆形”细胞的形态和生物学特性。结果表明,携带hTERT基因的从ES细胞分化来的圆形细胞在体外可大量增殖,持续传代,95％具有血管内皮细胞的一些特有标志和管道化生长特征。因此,通过人端粒酶基因的转染途径可解决由ES细胞诱导分化而来的内皮细胞扩增和永生化问题,为构建组织工程化血管及其它人工血管的内皮化提供种子细胞来源打下基础。     

Therapeutic potential of human-induced pluripotent stem cell-derived endothelial cells in a bleomycin-induced scleroderma mouse model

Vascular injury and destruction of endothelial cells (ECs) are the early events in scleroderma (SSc) patients. This study aims to investigate the therapeutic potential of human-induced pluripotent stem cell-derived ECs (hiPSC-ECs) to treat SSc. We have assessed the functional differentiation of hiPSC-ECs and compared them with human embryonic stem cell-derived ECs (hESC-ECs) by a variety of in vitro experimental approaches. Additionally, we evaluated the therapeutic potential of hiPSC-ECs in a bleomycin-induced SSc mouse model. Our results demonstrated that hiPSC-ECs and hESC-ECs showed similar maximum expressions of FLK1 (early EC marker) at day five during differentiation. After sorting and culturing, the FLK1-positive cells exhibited spindle and subsequent endothelial cobblestone morphology in EGM2 medium. The hESC-ECs and hiPSC-ECs also expressed late EC markers CD31 (68% and 75%), CD144 (50% and 61%), CD146 (46% and 61%), and DiI-labeled acetylated low-density lipoprotein (DiI-ac-LDL) uptake (55% and 63%), respectively. They additionally formed capillary-like structures on Matrigel. Analyses of the transplantation of sorted CD31-positive hiPSC-ECs into the bleomycin-induced SSc mouse model demonstrated that these cells participate in recovery of the damaged vessels. There was a reduction in collagen content; the number of total and degranulated mast cells returned to their normal state, and bleomycin-induced wounds as well as skin fibrosis improved four weeks after transplantation of hiPSC-ECs. Our findings have shown that the differentiation process from hESCs and hiPSCs to vascular cell components is similar. Additionally, this is the first study to determine the therapeutic potential of vascular cells from hiPSCs in the treatment of an SSc model. In the future, with further validation, these may be used as an appropriate source for the treatment of SSc patients.     

Remyelination induced by a DNA aptamer in a mouse model of multiple sclerosis

Multiple sclerosis (MS) is a debilitating inflammatory disease of the central nervous system (CNS) characterized by local destruction of the insulating myelin surrounding neuronal axons. With more than 200 million MS patients worldwide, the absence of treatments that prevent progression or induce repair poses a major challenge. Anti-inflammatory therapies have met with limited success only in preventing relapses. Previous screening of human serum samples revealed natural IgM antibodies that bind oligodendrocytes and promote both cell signaling and remyelination of CNS lesions in an MS model involving chronic infection of susceptible mice by Theiler's encephalomyelitis virus and in the lysolecithin model of focal demyelination. This intriguing result raises the possibility that molecules with binding specificity for oligodendrocytes or myelin components may promote therapeutic remyelination in MS. Because of the size and complexity of IgM antibodies, it is of interest to identify smaller myelin-specific molecules with the ability to promote remyelination in vivo. Here we show that a 40-nucleotide single-stranded DNA aptamer selected for affinity to murine myelin shows this property. This aptamer binds multiple myelin components in vitro. Peritoneal injection of this aptamer results in distribution to CNS tissues and promotes remyelination of CNS lesions in mice infected by Theiler's virus. Interestingly, the selected DNA aptamer contains guanosine-rich sequences predicted to induce folding involving guanosine quartet structures. Relative to monoclonal antibodies, DNA aptamers are small, stable, and non-immunogenic, suggesting new possibilities for MS treatment.     

Lanthionine ketimine ester provides benefit in a mouse model of multiple sclerosis

Lanthionine ketimine (LK) is a natural sulfur amino acid metabolite which binds to collapsin response mediator protein‐2 (CRMP2), an abundant brain protein that interacts with multiple partners to regulate microtubule dynamics, neurite growth and retraction, axonal transport, and neurotransmitter release. LK ethyl‐ester (LKE) is a cell‐permeable synthetic derivative that promotes neurogenesis, suppresses nitric oxide production from microglia, and reduces neurotoxicity of microglia‐conditioned medium. These properties led us to test the effects of LKE in experimental autoimmune encephalomyelitis (EAE), a commonly used mouse model of multiple sclerosis. Female C57Bl/6 mice were immunized with myelin oligodendrocyte glycoprotein peptide 35–55 to develop a chronic disease. LKE was provided in the chow at 100 ppm, ad libitum beginning when the mice reached moderate clinical signs. Over the following 4 weeks the LKE‐treated mice showed a significant reduction in clinical signs compared to vehicle‐treated mice. LKE dose dependently reduced IFNγ production from splenic T cells, but had no effect on IL‐17 production suggesting protective effects were mediated within the CNS. Electron microscopy revealed that, compared to sham mice, EAE mice had significant neurodegeneration in both the optic nerve and spinal cord, which was reduced in the LKE‐treated mice. In contrast only minimal disruption of myelin was observed at this time point. In the optic nerve, measurements of axon caliber and myelin thickness showed little changes between sham and EAE mice, however, treatment with LKE increased the percentage of axons with thicker myelin and with larger axon calibers. In the spinal cord, only smaller effects of LKE on myelin thickness were observed. The effects of LKE were associated with a reduced relative level of phosphorylated CRMP2 to CRMP2. Together, these results demonstrate that LKE reduces neurodegeneration in a chronic EAE model of MS, which could have translation potential for treatment of progressive forms of MS.

     

Embryonic stem cells and skin: from cellular model to therapeutic potential

Embryonic stem (ES) cells are pluripotent cells able to differentiate into many cell types in vitro, thus providing a potential unlimited supply of cells for cognitive in vitro studies and cell-based therapy. We recently reported their efficient ability to recapitulate ectodermal and epidermal fates and form, in culture, a multilayered epidermis coupled with an underlying dermal compartment, similar to native skin. Thus, ES cells have the potential to recapitulate the reciprocal instructive ectodermal-mesodermal commitments, characteristic of embryonic skin formation. We clarified the function of BMP-4 in the binary neuroectodermal choice by stimulating sox-1+ neural precursors to undergo specific apoptosis while inducing epidermal differentiation. We further demonstrated that p63 stimulates ectodermal cell proliferation and is necessary for epidermal commitment. We provided further evidence that this unique cellular model provides a powerful tool to identify the molecular mechanisms controlling normal skin development and to investigate human ectodermal dysplasia congenital pathologies linked to p63 (in p63-ectodermal dysplasia human congenital pathologies). Epidermal stem cell activity has been used for years to repair skin injuries, but ex vivo keratinocyte amplification has limitations and grafted skin homeostasis is not totally satisfactory. Human ES cells raise hopes that the understanding of developmental steps leading to the generation of epidermal stem cells will once be translated into therapeutic benefit. We recently demonstrated that human embryonic stem cells can give rise to a stable somatic ectodermal cell population. Its finite population doubling, normal cell cycle kinetics and the absence of teratoma formation strongly suggest that, although derived from human embryonic stem cells, these ectodermal cells represent a clinically safe somatic cell population. They could thus be particularly useful as a source for committed, homogeneous, non-tumorigenic cell populations to be employed in clinical trials for epithelial stem cell loss.     

Brain engraftment and therapeutic potential of stem/progenitor cells derived from mouse skin

Skin stem/progenitor cells (SKPs) derive from the dermis and in culture can generate mesodermal and neural progenies. To investigate their potential for the treatment of brain diseases, we first injected SKPs into the brain of syngeneic mice. Brain histology indicated that most SKPs remained undifferentiated and clustered at the injection site, while, in vitro, 17% of SKPs expressed neural markers, as assessed by flow cytometry. After labeling with magnetodendrimers, murine and human SKPs were detected by magnetic resonance imaging even 5 months after brain injection. To evaluate their therapeutic potential on malignant gliomas, IL-4 SKPs (i.e. SKPs transduced by a lentiviral vector carrying the cDNA of the anti-glioma cytokine interleukin-4) were injected into GL261 experimental gliomas. IL-4-SKPs prolonged significantly the survival of tumor-bearing mice: furthermore, GL261 gliomas attracted SKPs originally injected into the contralateral hemisphere. Thus, prolonged survival, capacity for transgene expression, and lack of uncontrolled proliferation suggest that SKPs warrant further consideration as therapeutic tools for brain tumors and, possibly, other neurological disorders.     

Effects of progesterone in the spinal cord of a mouse model of multiple sclerosis

The spinal cord is a target of progesterone (PROG), as demonstrated by the expression of intracellular and membrane PROG receptors and by its myelinating and neuroprotective effects in trauma and neurodegeneration. Here we studied PROG effects in mice with experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis characterized by demyelination and immune cell infiltration in the spinal cord. Female C57BL/6 mice were immunized with a myelin oligodendrocyte glycoprotein peptide (MOG_40–54). One week before EAE induction, mice received single pellets of PROG weighing either 20 or 100 mg or remained free of steroid treatment. On average, mice developed clinical signs of EAE 9–10 days following MOG administration. The spinal cord white matter of EAE mice showed inflammatory cell infiltration and circumscribed demyelinating areas, demonstrated by reductions of luxol fast blue (LFB) staining, myelin basic protein (MBP) and proteolipid protein (PLP) immunoreactivity (IR) and PLP mRNA expression. In motoneurons, EAE reduced the expression of the alpha 3 subunit of Na,K-ATPase mRNA. In contrast, EAE mice receiving PROG showed less inflammatory cell infiltration, recovery of myelin proteins and normal grain density of neuronal Na,K-ATPase mRNA. Clinically, PROG produced a moderate delay of disease onset and reduced the clinical scores. Thus, PROG attenuated disease severity, and reduced the inflammatory response and the occurrence of demyelination in the spinal cord during the acute phase of EAE.