A novel Monoclonal Antibody against Notch1 Targets Leukemia-associated Mutant Notch1 and Depletes Therapy Resistant Cancer Stem Cells in Solid Tumors

Higher Notch signaling is known to be associated with hematological and solid cancers. We developed a potential immunotherapeutic monoclonal antibody (MAb) specific for the Negative Regulatory Region of Notch1 (NRR). The MAb604.107 exhibited higher affinity for the ``Gain-offunction'' mutants of Notch1 NRR associated with T Acute lymphoblastic Leukemia (T-ALL). Modeling of the mutant NRR with 12 amino-acid insertion demonstrated ``opening'' resulting in exposure of the S2-cleavage site leading to activated Notch1 signaling. The MAb, at low concentrations (1-2 mu g/ml), inhibited elevated ligand-independent Notch1 signaling of NRR mutants, augmented effect of Thapsigargin, an inhibitor of mutant Notch1, but had no effect on the wild-type Notch1. The antibody decreased proliferation of the primary T-ALL cells and depleted leukemia initiating CD34/CD44 high population. At relatively high concentrations, (10-20 mu g/ml), the MAb affected Notch1 signaling in the breast and colon cancer cell lines. The Notch-high cells sorted from solid-tumor cell lines exhibited characteristics of cancer stem cells, which were inhibited by the MAb. The antibody also increased the sensitivity to Doxorubucinirubicin. Further, the MAb impeded the growth of xenografts from breast and colon cancer cells potentiated regression of the tumors along with Doxorubucin. Thus, this antibody is potential immunotherapeutic tool for different cancers.

MicroRNA-132 is a physiological regulator of hematopoietic stem cell function and B-cell development

MicroRNAs are a class of small non-coding RNAs that negatively regulate gene expression. Several microRNAs have been implicated in altering hematopoietic cell fate decisions. Importantly, deregulation of many microRNAs can lead to deleterious consequences in the hematopoietic system, including the onset of cancer, autoimmunity, or a failure to respond effectively to infection. As such, microRNAs fine-tune the balance between normal hematopoietic output and pathologic consequences. In this work, we explore the role of two microRNAs, miR-132 and miR-125b, in regulating hematopoietic stem cell (HSC) function and B cell development. In particular, we uncover the role of miR-132 in maintaining the appropriate balance between self-renewal, differentiation, and survival in aging HSCs by buffering the expression of a critical transcription factor, FOXO3. By maintain this balance, miR-132 may play a critical role in preventing aging-associated hematopoietic conditions such as autoimmune disease and cancer. We also find that miR-132 plays a critical role in B cell development by targeting a key transcription factor, Sox4, that is responsible for the differentiation of pro-B cells into pre-B cells. We find that miR-132 regulates B cell apoptosis, and by delivering miR-132 to mice that are predisposed to developing B cell cancers, we can inhibit the formation of these cancers and improve the survival of these mice. In addition to miR-132, we uncovered the role of another critical microRNA, miR-125b, that potentiates hematopoietic stem cell function. We found that enforced expression of miR-125b causes an aggressive myeloid leukemia by downregulation of its target Lin28a. Importantly, miR-125b also plays a critical role in inhibiting the formation of pro-B cells. Thus, we have discovered two microRNAs with important roles in regulating normal hematopoiesis, and whose dregulation can lead to deleterious consequences such as cancer in the aging hematopoietic system. Both miR-132 and miR-125b may therefore be targeted for therapeutics to inhibit age-related immune diseases associated with the loss of HSC function and cancer progression.

Transplantable neural progenitor populations derived from Rhesus monkey embryonic stem cells

Cell-based therapies using embryonic stem cells (ESCs) in the treatment of neural disease will require the generation of homogenous donor neural progenitor (NP) populations. Here we describe an efficient culture system containing hepatocyte growth factor (HGF) and G5 supplement for the production of highly enriched (88.3% +/- 8.1%)populations of NPs from rhesus monkey ESCs. Additional purification resulted in NP preparations that were 98% nestin positive. Moreover, NPs, as monolayers or neurospheres, could be maintained for prolonged periods of time in media containing HGF+G5 or G5 alone. In vitro differentiation and in vivo transplantation assays showed that NPs could differentiate into neurons, astrocytes, and oligodendrocytes. The kinds and quantities of differentiated cells derived from NPs were closely correlated with their niches in vivo. Glial differentiation was predominant in periventricular areas, whereas cells migrating into the cortex were mostly neurons. Cell counts showed that 2 months after transplantation, approximately 25% of transplanted NPs survived and 65% - 80% of the surviving transplanted cells migrated along the ventricular wall or in a radial fashion. Subcloning demonstrated that several clonal lines derived from NPs expressed nestin and differentiated into three neural lineages in vitro and in rat brains in vivo. In contrast, some subcloned lines showed restricted differentiation both in vitro and in vivo in rat brains. These observations set the stage for obtaining highly enriched NPs and evaluating the efficacy of NP-based transplantation therapy in the nonhuman primate and will provide a platform for probing the molecular mechanisms that control neural induction.

Dissecting Signaling Pathways That Govern Self-renewal of Rabbit Embryonic Stem Cells

The pluripotency and self-renewal of embryonic stem cells (ESC) are regulated by a variety of cytokines/growth factors with some species differences. We reported previously that rabbit ESC (rESC) are more similar to primate ESC than to mouse ESC. However,

Neural progenitors derived from monkey embryonic stem cells in a simple monoculture system

A simple monoculture system. combined with a chemically defined medium containing hepatocyte growth factor (HGF) and G5 supplement, was used to induce rhesus monkey embryonic stem cells (rESC) directly into neuroepithelial (NE) cells. Under these conditio

Differentiated adipose-derived stem cells act synergistically with RGD-modified surfaces to improve neurite outgrowth in a co-culture model.

Peripheral nerve damage is a problem encountered after trauma and during surgery and the development of synthetic polymer conduits may offer a promising alternative to autografts. In order to improve the performance of the polymer to be used for nerve conduits, poly-ε-caprolactone (PCL) films were chemically functionalized with RGD moieties, using a chemical reaction previously developed. In vitro cultures of dissociated dorsal root ganglion (DRG) neurons provide a valid model to study different factors affecting axonal growth. In this work, DRG neurons were cultured on RGD-functionalized PCL films. Adult adipose-derived stem cells differentiated to Schwann cells (dASCs) were initially cultured on the functionalized PCL films, resulting in improved attachment and proliferation. dASCs were also co-cultured with DRG neurons on treated and untreated PCL to assess stimulation by dASCs on neurite outgrowth. Neuron response was generally poor on untreated PCL films, but long neurites were observed in the presence of dASCs or RGD moieties. A combination of the two factors enhanced even further neurite outgrowth, acting synergistically. Finally, in order to better understand the extracellular matrix (ECM)-cell interaction, a β1 integrin blocking experiment was carried out. Neurite outgrowth was not affected by the specific antibody blocking, showing that β1 integrin function can be compensated by other molecules present on the cell membrane. Copyright © 2013 John Wiley & Sons, Ltd.

Ligament-derived matrix stimulates a ligamentous phenotype in human adipose-derived stem cells.

Human adipose stem cells (hASCs) can differentiate into a variety of phenotypes. Native extracellular matrix (e.g., demineralized bone matrix or small intestinal submucosa) can influence the growth and differentiation of stem cells. The hypothesis of this study was that a novel ligament-derived matrix (LDM) would enhance expression of a ligamentous phenotype in hASCs compared to collagen gel alone. LDM prepared using phosphate-buffered saline or 0.1% peracetic acid was mixed with collagen gel (COL) and was evaluated for its ability to induce proliferation, differentiation, and extracellular matrix synthesis in hASCs over 28 days in culture at different seeding densities (0, 0.25 x 10(6), 1 x 10(6), or 2 x 10(6) hASC/mL). Biochemical and gene expression data were analyzed using analysis of variance. Fisher's least significant difference test was used to determine differences between treatments following analysis of variance. hASCs in either LDM or COL demonstrated changes in gene expression consistent with ligament development. hASCs cultured with LDM demonstrated more dsDNA content, sulfated-glycosaminoglycan accumulation, and type I and III collagen synthesis, and released more sulfated-glycosaminoglycan and collagen into the medium compared to hASCs in COL (p

Nanotopography-induced changes in focal adhesions, cytoskeletal organization, and mechanical properties of human mesenchymal stem cells.

The growth of stem cells can be modulated by physical factors such as extracellular matrix nanotopography. We hypothesize that nanotopography modulates cell behavior by changing the integrin clustering and focal adhesion (FA) assembly, leading to changes in cytoskeletal organization and cell mechanical properties. Human mesenchymal stem cells (hMSCs) cultured on 350 nm gratings of tissue-culture polystyrene (TCPS) and polydimethylsiloxane (PDMS) showed decreased expression of integrin subunits alpha2, alpha , alpha V, beta2, beta 3 and beta 4 compared to the unpatterned controls. On gratings, the elongated hMSCs exhibited an aligned actin cytoskeleton, while on unpatterned controls, spreading cells showed a random but denser actin cytoskeleton network. Expression of cytoskeleton and FA components was also altered by the nanotopography as reflected in the mechanical properties measured by atomic force microscopy (AFM) indentation. On the rigid TCPS, hMSCs on gratings exhibited lower instantaneous and equilibrium Young's moduli and apparent viscosity. On the softer PDMS, the effects of nanotopography were not significant. However, hMSCs cultured on PDMS showed lower cell mechanical properties than those on TCPS, regardless of topography. These suggest that both nanotopography and substrate stiffness could be important in determining mechanical properties, while nanotopography may be more dominant in determining the organization of the cytoskeleton and FAs.

Diet-induced obesity alters the differentiation potential of stem cells isolated from bone marrow, adipose tissue and infrapatellar fat pad: the effects of free fatty acids.

INTRODUCTION: Obesity is a major risk factor for several musculoskeletal conditions that are characterized by an imbalance of tissue remodeling. Adult stem cells are closely associated with the remodeling and potential repair of several mesodermally derived tissues such as fat, bone and cartilage. We hypothesized that obesity would alter the frequency, proliferation, multipotency and immunophenotype of adult stem cells from a variety of tissues. MATERIALS AND METHODS: Bone marrow-derived mesenchymal stem cells (MSCs), subcutaneous adipose-derived stem cells (sqASCs) and infrapatellar fat pad-derived stem cells (IFP cells) were isolated from lean and high-fat diet-induced obese mice, and their cellular properties were examined. To test the hypothesis that changes in stem cell properties were due to the increased systemic levels of free fatty acids (FFAs), we further investigated the effects of FFAs on lean stem cells in vitro. RESULTS: Obese mice showed a trend toward increased prevalence of MSCs and sqASCs in the stromal tissues. While no significant differences in cell proliferation were observed in vitro, the differentiation potential of all types of stem cells was altered by obesity. MSCs from obese mice demonstrated decreased adipogenic, osteogenic and chondrogenic potential. Obese sqASCs and IFP cells showed increased adipogenic and osteogenic differentiation, but decreased chondrogenic ability. Obese MSCs also showed decreased CD105 and increased platelet-derived growth factor receptor α expression, consistent with decreased chondrogenic potential. FFA treatment of lean stem cells significantly altered their multipotency but did not completely recapitulate the properties of obese stem cells. CONCLUSIONS: These findings support the hypothesis that obesity alters the properties of adult stem cells in a manner that depends on the cell source. These effects may be regulated in part by increased levels of FFAs, but may involve other obesity-associated cytokines. These findings contribute to our understanding of mesenchymal tissue remodeling with obesity, as well as the development of autologous stem cell therapies for obese patients.

Tissue engraftment of hypoxic-preconditioned adipose-derived stem cells improves flap viability.

Adipose-derived stem cells (ASCs) have the ability to release multiple growth factors in response to hypoxia. In this study, we investigated the potential of ASCs to prevent tissue ischemia. We found conditioned media from hypoxic ASCs had increased levels of vascular endothelial growth factor (VEGF) and enhanced endothelial cell tubule formation. To investigate the effect of injecting rat ASCs into ischemic flaps, 21 Lewis rats were divided into three groups: control, normal oxygen ASCs (10(6) cells), and hypoxic preconditioned ASCs (10(6) cells). At the time of flap elevation, the distal third of the flap was injected with the treatment group. At 7 days post flap elevation, flap viability was significantly improved with injection of hypoxic preconditioned ASCs. Cluster of differentiation-31-positive cells were more abundant along the margins of flaps injected with ASCs. Fluorescent labeled ASCs localized aside blood vessels or throughout the tissue, dependent on oxygen preconditioning status. Next, we evaluated the effect of hypoxic preconditioning on ASC migration and chemotaxis. Hypoxia did not affect ASC migration on scratch assay or chemotaxis to collagen and laminin. Thus, hypoxic preconditioning of injected ASCs improves flap viability likely through the effects of VEGF release. These effects are modest and represent the limitations of cellular and growth factor-induced angiogenesis in the acute setting of ischemia.

Bioreactor Expansion of Human Adult Bone Marrow-Derived Mesenchymal Stem Cells

Supplementation of mesenchymal stem cells (MSCs) during hematopoietic stem cell transplantation (HSCT) alleviates complications such as graft-versus-host disease, leading to a speedy recovery of hematopoiesis. To meet such clinical demand, a fast MSCs expansion method is required. In the present study, we examined the feasibility of expanding MSCs from the isolated bone marrow mononuclear cells using a rotary bioreactor system. The cells were cultured in a rotary bioreactor with Myelocult� medium containing a combination of supplementary factors, including stem cell factor (SCF), interleukin 3 and 6 (IL-3, IL-6). After 8 days of culture, total cell numbers, Stro-1+CD44+CD34- MSCs and CD34+CD44+Stro-1- HSCs were increased 9, 29, and 8 folds respectively. Colony forming efficiency-fibroblast per day (CFE-F/day) of the bioreactor-treated cells was 1.44-fold higher than that of the cells without bioreactor treatment. The bioreactor-expanded MSCs showed expression of primitive MSCs markers endoglin (SH2) and vimentin, whereas markers associated with lineage differentiation including osteocalcin (osteogenesis), Type II collagen (chondrogenesis) and C/EBPα (adipogenesis) were not detected. Upon induction, the bioreactor-expanded MSCs were able to differentiate into osteoblasts, chondrocytes and adipocytes. Taken together, we conclude that the rotary bioreactor with the modified Myelocult� medium reported in this study may be used to rapidly expand MSCs.

Isolation of retinal progenitor and stem cells from the porcine eye

Purpose: Retinal progenitor cells (RPCs) and retinal stem cells (RSCs) from rodents and humans have been isolated and characterized in vitro. Transplantation experiments have confirmed their potential as tools for cell replacement in retinal degenerative diseases. The pig represents an ideal pre-clinical animal model to study the impact of transplantation because of the similarity of its eye to the human eye. However, little is known about porcine RPCs and RSCs. We aimed to identify and characterize in vitro RPCs and RSCs from porcine ocular tissues. Methods: Cells from different subregions of embryonic, postnatal and adult porcine eyes were grown in suspension sphere culture in serum-free medium containing basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). Growth curves and BrdU incorporation assays were performed to establish the proliferative capacity of isolated porcine retina-derived RPCs and ciliary epithelium (CE)-derived RSCs. Self-renewal potential was investigated by subsphere formation assays. Changes in gene expression were assayed by reverse transcription polymerase chain reaction (RT-PCR) at different passages in culture. Finally, differentiation was induced by addition of serum to the cultures and expression of markers for retinal cell types was detected by immunohistochemical staining with specific antibodies. Results: Dissociated cells from embryonic retina and CE at different postnatal ages generated primary nestin- and Pax6-immunoreactive neurosphere colonies in vitro in numbers that decreased with age. Embryonic and postnatal retina-derived RPCs and young CE-derived RSCs displayed self-renewal capacity, generating secondary neurosphere colonies. However, their self-renewal and proliferation capacity gradually decreased and they became more committed to differentiated states with subsequent passages. The expansion capacity of RPCs and RSCs was higher when they were maintained in monolayer culture. Porcine RPCs and RSCs could be induced to differentiate in vitro to express markers of retinal neurons and glia. Conclusions: Porcine retina and CE contain RPCs and RSCs which are undifferentiated, self-renewing and multipotent and which show characteristics similar to their human counterparts. Therefore, the pig could be a useful source of cells to further investigate the cell biology of RPCs and RSCs and it could be used as a non-primate large animal model for pre-clinical studies on stem cell-based approaches to regenerative medicine in the retina.

Adult neural stem cells expressing IL-10 confer potent immunomodulation and remyelination in experimental autoimmune encephalitis

Adult neural stem cells (aNSCs) derived from the subventricular zone of the brain show therapeutic effects in EAE, an animal model of the chronic inflammatory neurodegenerative disease MS; however, the beneficial effects are modest. One critical weakness of aNSC therapy may be an insufficient antiinflammatory effect. Here, we demonstrate that i.v. or i.c.v. injection of aNSCs engineered to secrete IL-10 (IL-10–aNSCs), a potent immunoregulatory cytokine, induced more profound functional and pathological recovery from ongoing EAE than that with control aNSCs. IL-10–aNSCs exhibited enhanced antiinflammatory effects in the periphery and inflammatory foci in the CNS compared with control aNSCs, more effectively reducing myelin damage, a hallmark of MS. When compared with mice treated with control aNSCs, those treated with IL-10–aNSCs demonstrated differentiation of transplanted cells into greater numbers of oligodendrocytes and neurons but fewer astrocytes, thus enhancing exogenous remyelination and neuron/axonal growth. Finally, IL-10–aNSCs converted a hostile environment to one supportive of neurons/oligodendrocytes, thereby promoting endogenous remyelination. Thus, aNSCs engineered to express IL-10 show enhanced ability to induce immune suppression, remyelination, and neuronal repair and may represent a novel approach that can substantially improve the efficacy of neural stem cell–based therapy in EAE/MS.

Vascular stem cells and ischaemic retinopathies

Retinal ischaemic disorders such as diabetic retinopathy and retinal vein occlusion are common. The hypoxia-related stimuli from oxygen-deprived neural and glial networks can drive expression of growth factors and cytokines which induce leakage from the surviving vasculature and/or pre-retinal and papillary neovascularisation. If left untreated, retinal vascular stasis, hypoxia or ischaemia can lead to macular oedema or fibro-vascular scar formation which are associated with severe visual impairment, and even blindness. Current therapies for ischaemic retinopathies include laser photocoagulation, injection of corticosteroids or VEGF-antibodies and vitreoretinal surgery, however they carry significant side effects. As an alternative approach, we propose that if reparative intra-retinal angiogenesis can be harnessed at the appropriate stage, ischaemia could be contained or reversed. This review provides evidence that reperfusion of ischaemic retina and suppression of sight-threatening sequelae is possible in both experimental and clinical settings. In particular, there is emphasis on the clinical potential for endothelial progenitor cells (EPCs) to promote vascular repair and reversal of ischaemic injury in various tissues including retina. Gathering evidence from an extensive published literature, we outline the molecular and phenotypic nature of EPCs, how they are altered in disease and provide a rationale for harnessing the vascular reparative properties of various cell sub-types. When some of the remaining questions surrounding the clinical use of EPCs are addressed, they may provide an exciting new therapeutic option for treating ischaemic retinopathies. (C) 2011 Elsevier Ltd. All rights reserved.

Therapeutic Effects of Human Mesenchymal Stem Cells in Ex Vivo Human Lungs Injured with Live Bacteria

Rationale: Mesenchymal stem cells secrete paracrine factors that can regulate lung permeability and decrease inflammation, making it a potentially attractive therapy for acute lung injury. However, concerns exist whether mesenchymal stem cells' immunomodulatory properties may have detrimental effects if targeted toward infectious causes of lung injury. Objectives: Therefore, we tested the effect of mesenchymal stem cells on lung fluid balance, acute inflammation, and bacterial clearance. Methods: We developed an Escherichia coli pneumonia model in our ex vivo perfused human lung to test the therapeutic effects of mesenchymal stem cells on bacterial-induced acute lung injury. Measurements and Main Results: Clinical-grade human mesenchymal stem cells restored alveolar fluid clearance to a normal level, decreased inflammation, and were associated with increased bacterial killing and reduced bacteremia, in part through increased alveolar macrophage phagocytosis and secretion of antimicrobial factors. Keratinocyte growth factor, a soluble factor secreted by mesenchymal stem cells, duplicated most of the antimicrobial effects. In subsequent in vitro studies, we discovered that human monocytes expressed the keratinocyte growth factor receptor, and that keratinocyte growth factor decreased apoptosis of human monocytes through AKT phosphorylation, an effect that increased bacterial clearance. Inhibition of keratinocyte growth factor by a neutralizing antibody reduced the antimicrobial effects of mesenchymal stem cells in the ex vivo perfused human lung and monocytes grown in vitro injured with E. coli bacteria. Conclusions: In E. coli-injured human lungs, mesenchymal stem cells restored alveolar fluid clearance, reduced inflammation, and exerted antimicrobial activity, in part through keratinocyte growth factor secretion.