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.

Directed differentiation and functional maturation of cortical interneurons from human embryonic stem cells

Human pluripotent stem cells are a powerful tool for modeling brain development and disease. The human cortex is composed of two major neuronal populations: projection neurons and local interneurons. Cortical interneurons comprise a diverse class of cell types expressing the neurotransmitter GABA. Dysfunction of cortical interneurons has been implicated in neuropsychiatric diseases, including schizophrenia, autism, and epilepsy. Here, we demonstrate the highly efficient derivation of human cortical interneurons in an NKX2.1::GFP human embryonic stem cell reporter line. Manipulating the timing of SHH activation yields three distinct GFP+ populations with specific transcriptional profiles, neurotransmitter phenotypes, and migratory behaviors. Further differentiation in a murine cortical environment yields parvalbumin- and somatostatin-expressing neurons that exhibit synaptic inputs and electrophysiological properties of cortical interneurons. Our study defines the signals sufficient for modeling human ventral forebrain development in vitro and lays the foundation for studying cortical interneuron involvement in human disease pathology.

HGF Expressing Stem Cells in Usual Interstitial Pneumonia Originate from the Bone Marrow and Are Antifibrotic

BACKGROUND Pulmonary fibrosis may result from abnormal alveolar wound repair after injury. Hepatocyte growth factor (HGF) improves alveolar epithelial wound repair in the lung. Stem cells were shown to play a major role in lung injury, repair and fibrosis. We studied the presence, origin and antifibrotic properties of HGF-expressing stem cells in usual interstitial pneumonia. METHODS Immunohistochemistry was performed in lung tissue sections and primary alveolar epithelial cells obtained from patients with usual interstitial pneumonia (UIP, n = 7). Bone marrow derived stromal cells (BMSC) from adult male rats were transfected with HGF, instilled intratracheally into bleomycin injured rat lungs and analyzed 7 and 14 days later. RESULTS In UIP, HGF was expressed in specific cells mainly located in fibrotic areas close to the hyperplastic alveolar epithelium. HGF-positive cells showed strong co-staining for the mesenchymal stem cell markers CD44, CD29, CD105 and CD90, indicating stem cell origin. HGF-positive cells also co-stained for CXCR4 (HGF+/CXCR4+) indicating that they originate from the bone marrow. The stem cell characteristics were confirmed in HGF secreting cells isolated from UIP lung biopsies. In vivo experiments showed that HGF-expressing BMSC attenuated bleomycin induced pulmonary fibrosis in the rat, indicating a beneficial role of bone marrow derived, HGF secreting stem cells in lung fibrosis. CONCLUSIONS HGF-positive stem cells are present in human fibrotic lung tissue (UIP) and originate from the bone marrow. Since HGF-transfected BMSC reduce bleomycin induced lung fibrosis in the bleomycin lung injury and fibrosis model, we assume that HGF-expressing, bone-marrow derived stem cells in UIP have antifibrotic properties.

Anaplastic oligodendroglioma arising from the brain stem and featuring 1p/19q co-deletion

With respect to localization, oligodendrogliomas are characterized by a marked preponderance of the cerebral hemispheres. Outside these typical sites, any tumor histopathologically reminiscent of oligodendroglioma a priori is likely to represent one of its morphological mimics, including clear cell ependymoma, neurocytoma, pilocytic astrocytoma or glioneuronal tumors. This is particularly relevant as several of the latter are in principle curable by surgery. Among extrahemispherical sites, bona fide oligodendroglioma - as characterized by loss of heterozygosity (LOH) of chromosome arms 1p and 19q - so far has not been documented to occur in the brain stem. Here, we report the case of a 55-year-old female patient with an anaplastic oligodendroglioma (WHO grade III) of the brain stem and cerebellum diagnosed by stereotactic biopsy and featuring combined LOH of 1p and 19q. A morphological peculiarity was a population of interspersed tumor giant cells, a phenomenon that has been referred to as polymorphous oligodendroglioma. Our findings confirm the notion that - although very infrequently - true oligodendrogliomas do occur in the infratentorial compartment.

In vitro fabrication of autologous living tissue-engineered vascular grafts based on prenatally harvested ovine amniotic fluid-derived stem cells

Amniotic fluid cells (AFCs) have been proposed as a valuable source for tissue engineering and regenerative medicine. However, before clinical implementation, rigorous evaluation of this cell source in clinically relevant animal models accepted by regulatory authorities is indispensable. Today, the ovine model represents one of the most accepted preclinical animal models, in particular for cardiovascular applications. Here, we investigate the isolation and use of autologous ovine AFCs as cell source for cardiovascular tissue engineering applications. Fetal fluids were aspirated in vivo from pregnant ewes (n = 9) and from explanted uteri post mortem at different gestational ages (n = 91). Amniotic non-allantoic fluid nature was evaluated biochemically and in vivo samples were compared with post mortem reference samples. Isolated cells revealed an immunohistochemical phenotype similar to ovine bone marrow-derived mesenchymal stem cells (MSCs) and showed expression of stem cell factors described for embryonic stem cells, such as NANOG and STAT-3. Isolated ovine amniotic fluid-derived MSCs were screened for numeric chromosomal aberrations and successfully differentiated into several mesodermal phenotypes. Myofibroblastic ovine AFC lineages were then successfully used for the in vitro fabrication of small- and large-diameter tissue-engineered vascular grafts (n = 10) and cardiovascular patches (n = 34), laying the foundation for the use of this relevant pre-clinical in vivo assessment model for future amniotic fluid cell-based therapeutic applications. Copyright © 2013 John Wiley & Sons, Ltd.

Inducible caspase 9 suicide gene to improve the safety of allodepleted T cells after haploidentical stem cell transplantation.

Addback of donor T cells following T cell-depleted stem cell transplantation (SCT) can accelerate immune reconstitution and be effective against relapsed malignancy. After haploidentical SCT, a high risk of graft-versus-host disease (GVHD) essentially precludes this option, unless the T cells are first depleted of alloreactive precursor cells. Even then, the risks of severe GVHD remain significant. To increase the safety of the approach and thereby permit administration of larger T cell doses, we used a suicide gene, inducible caspase 9 (iCasp9), to transduce allodepleted T cells, permitting their destruction should administration have adverse effects. We made a retroviral vector encoding iCasp9 and a selectable marker (truncated CD19). Even after allodepletion (using anti-CD25 immunotoxin), donor T cells could be efficiently transduced, expanded, and subsequently enriched by CD19 immunomagnetic selection to >90% purity. These engineered cells retained antiviral specificity and functionality, and contained a subset with regulatory phenotype and function. Activating iCasp9 with a small-molecule dimerizer rapidly produced >90% apoptosis. Although transgene expression was downregulated in quiescent T cells, iCasp9 remained an efficient suicide gene, as expression was rapidly upregulated in activated (alloreactive) T cells. We have demonstrated the clinical feasibility of this approach after haploidentical transplantation by scaling up production using clinical grade materials.

ELUCIDATING THE ROLE OF CD44 EXPRESSION ON MESENCHYMAL STEM CELLS WITHIN THE TUMOR MICROENVIRONMENT

The tumor microenvironment is comprised of a vast array of heterogeneous cells including both normal and neoplastic cells. The tumor stroma recruitment process has been exploited for an effective gene delivery technique using bone marrow derived MSC. Targeted migration of the MSC toward the tumor microenvironment, while successful, is not yet fully understood. This study was designed to assess the role of CD44 in the migration of MSC toward the tumor microenvironment and to determine the implications of CD44-deficient MSC within the tumor stroma. Inhibition of MSC migration was evaluated through a variety of methods in vitro and in vivo including CD44 receptor knockdown, CD44 antagonists, CD44 neutralizing antibodies and small molecule inhibitor of matrix metalloproteinases. Blocking CD44 signaling through MMP inhibition was characterized by lack of intracellular domain cleavage and lead to the decrease in Twist gene expression. A functional relationship between CD44 and Twist expression was confirmed by chromatin immunoprecipitation. Next, a series of murine tumor models were used to examine the role of CD44 deficient stroma within the tumor microenvironment. Labeled transgenic CD44 knockout (KO) MSC or wild type (WT) C57/B6 MSC were used to analyze the stromal incorporation within murine breast carcinomas (EO771 and 4T1). Subsequent tumors were analyzed for vessel formation (CD31), and the presence of tumor associated fibroblast (TAF) markers, α-smooth muscle actin (α-SMA), fibroblast activation protein (FAP), and fibroblast specific protein (FSP). The tumors with CD44KO MSC cells had less vessel formation than the tumors with WT MSC. The lack of fibroblastic TAF population as defined by FAP/FSP expression by the CD44KO MSC admixed tumors suggest that the bone marrow derived population of MSC were unable to contribute to the fibroblastic stromal population. Subsequently, a bone marrow transplantation experiment confirmed the endogenous migratory deficiencies of the CD44KO bone marrow derived stromal cells toward the tumor microenvironment in vivo. WT mice with CD44KO bone marrow had less CD44KOderived tumor stroma compared to mice with WT bone marrow. These results indicate that CD44 is crucial to stromal cell migration and incorporation to the tumor microenvironment as TAF.

Abnormal expression of REST/NRSF and Myc in neural stem/progenitor cells causes cerebellar tumors by blocking neuronal differentiation.

Medulloblastoma, one of the most malignant brain tumors in children, is thought to arise from undifferentiated neural stem/progenitor cells (NSCs) present in the external granule layer of the cerebellum. However, the mechanism of tumorigenesis remains unknown for the majority of medulloblastomas. In this study, we found that many human medulloblastomas express significantly elevated levels of both myc oncogenes, regulators of neural progenitor proliferation, and REST/NRSF, a transcriptional repressor of neuronal differentiation genes. Previous studies have shown that neither c-Myc nor REST/NRSF alone could cause tumor formation. To determine whether c-Myc and REST/NRSF act together to cause medulloblastomas, we used a previously established cell line derived from external granule layer stem cells transduced with activated c-myc (NSC-M). These immortalized NSCs were able to differentiate into neurons in vitro. In contrast, when the cells were engineered to express a doxycycline-regulated REST/NRSF transgene (NSC-M-R), they no longer underwent terminal neuronal differentiation in vitro. When injected into intracranial locations in mice, the NSC-M cells did not form tumors either in the cerebellum or in the cerebral cortex. In contrast, the NSC-M-R cells did produce tumors in the cerebellum, the site of human medulloblastoma formation, but not when injected into the cerebral cortex. Furthermore, the NSC-M-R tumors were blocked from terminal neuronal differentiation. In addition, countering REST/NRSF function blocked the tumorigenic potential of NSC-M-R cells. To our knowledge, this is the first study in which abnormal expression of a sequence-specific DNA-binding transcriptional repressor has been shown to contribute directly to brain tumor formation. Our findings indicate that abnormal expression of REST/NRSF and Myc in NSCs causes cerebellum-specific tumors by blocking neuronal differentiation and thus maintaining the "stemness" of these cells. Furthermore, these results suggest that such a mechanism plays a role in the formation of human medulloblastoma.

REST maintains self-renewal and pluripotency of embryonic stem cells.

The neuronal repressor REST (RE1-silencing transcription factor; also called NRSF) is expressed at high levels in mouse embryonic stem (ES) cells, but its role in these cells is unclear. Here we show that REST maintains self-renewal and pluripotency in mouse ES cells through suppression of the microRNA miR-21. We found that, as with known self-renewal markers, the level of REST expression is much higher in self-renewing mouse ES cells than in differentiating mouse ES (embryoid body, EB) cells. Heterozygous deletion of Rest (Rest+/-) and its short-interfering-RNA-mediated knockdown in mouse ES cells cause a loss of self-renewal-even when these cells are grown under self-renewal conditions-and lead to the expression of markers specific for multiple lineages. Conversely, exogenously added REST maintains self-renewal in mouse EB cells. Furthermore, Rest+/- mouse ES cells cultured under self-renewal conditions express substantially reduced levels of several self-renewal regulators, including Oct4 (also called Pou5f1), Nanog, Sox2 and c-Myc, and exogenously added REST in mouse EB cells maintains the self-renewal phenotypes and expression of these self-renewal regulators. We also show that in mouse ES cells, REST is bound to the gene chromatin of a set of miRNAs that potentially target self-renewal genes. Whereas mouse ES cells and mouse EB cells containing exogenously added REST express lower levels of these miRNAs, EB cells, Rest+/- ES cells and ES cells treated with short interfering RNA targeting Rest express higher levels of these miRNAs. At least one of these REST-regulated miRNAs, miR-21, specifically suppresses the self-renewal of mouse ES cells, corresponding to the decreased expression of Oct4, Nanog, Sox2 and c-Myc. Thus, REST is a newly discovered element of the interconnected regulatory network that maintains the self-renewal and pluripotency of mouse ES cells.

Intravenous mesenchymal stem cell therapy for traumatic brain injury.

OBJECT: Cell therapy has shown preclinical promise in the treatment of many diseases, and its application is being translated to the clinical arena. Intravenous mesenchymal stem cell (MSC) therapy has been shown to improve functional recovery after traumatic brain injury (TBI). Herein, the authors report on their attempts to reproduce such observations, including detailed characterizations of the MSC population, non-bromodeoxyuridine-based cell labeling, macroscopic and microscopic cell tracking, quantification of cells traversing the pulmonary microvasculature, and well-validated measurement of motor and cognitive function recovery. METHODS: Rat MSCs were isolated, expanded in vitro, immunophenotyped, and labeled. Four million MSCs were intravenously infused into Sprague-Dawley rats 24 hours after receiving a moderate, unilateral controlled cortical impact TBI. Infrared macroscopic cell tracking was used to identify cell distribution. Immunohistochemical analysis of brain and lung tissues 48 hours and 2 weeks postinfusion revealed transplanted cells in these locations, and these cells were quantified. Intraarterial blood sampling and flow cytometry were used to quantify the number of transplanted cells reaching the arterial circulation. Motor and cognitive behavioral testing was performed to evaluate functional recovery. RESULTS: At 48 hours post-MSC infusion, the majority of cells were localized to the lungs. Between 1.5 and 3.7% of the infused cells were estimated to traverse the lungs and reach the arterial circulation, 0.295% reached the carotid artery, and a very small percentage reached the cerebral parenchyma (0.0005%) and remained there. Almost no cells were identified in the brain tissue at 2 weeks postinfusion. No motor or cognitive functional improvements in recovery were identified. CONCLUSIONS: The intravenous infusion of MSCs appeared neither to result in significant acute or prolonged cerebral engraftment of cells nor to modify the recovery of motor or cognitive function. Less than 4% of the infused cells were likely to traverse the pulmonary microvasculature and reach the arterial circulation, a phenomenon termed the "pulmonary first-pass effect," which may limit the efficacy of this therapeutic approach. The data in this study contradict the findings of previous reports and highlight the potential shortcomings of acute, single-dose, intravenous MSC therapy for TBI.