Here, we describe the multiple lentiviral expression (MuLE) system that allows multiple genetic alterations to be introduced simultaneously into mammalian cells. We created a toolbox of MuLE vectors that constitute a flexible, modular system for the rapid engineering of complex polycistronic lentiviruses, allowing combinatorial gene overexpression, gene knockdown, Cre-mediated gene deletion, or CRISPR/Cas9-mediated (where CRISPR indicates clustered regularly interspaced short palindromic repeats) gene mutation, together with expression of fluorescent or enzymatic reporters for cellular assays and animal imaging. Examples of tumor engineering were used to illustrate the speed and versatility of performing combinatorial genetics using the MuLE system. By transducing cultured primary mouse cells with single MuLE lentiviruses, we engineered tumors containing up to 5 different genetic alterations, identified genetic dependencies of molecularly defined tumors, conducted genetic interaction screens, and induced the simultaneous CRISPR/Cas9-mediated knockout of 3 tumor-suppressor genes. Intramuscular injection of MuLE viruses expressing oncogenic
Joachim Albers, Claudia Danzer, Markus Rechsteiner, Holger Lehmann, Laura P. Brandt, Tomas Hejhal, Antonella Catalano, Philipp Busenhart, Ana Filipa Gonçalves, Simone Brandt, Peter K. Bode, Beata Bode-Lesniewska, Peter J. Wild, Ian J. Frew
Pluripotent stem cells (PSCs) represent an alternative hematopoietic stem cell (HSC) source for treating hematopoietic disease. The limited engraftment of human PSC–derived (hPSC-derived) multipotent progenitor cells (MPP) has hampered the clinical application of these cells and suggests that MPP require additional cues for definitive hematopoiesis. We hypothesized that the presence of a vascular niche that produces Notch ligands jagged-1 (JAG1) and delta-like ligand-4 (DLL4) drives definitive hematopoiesis. We differentiated hes2 human embryonic stem cells (hESC) and
Jennifer L. Gori, Jason M. Butler, Yan-Yi Chan, Devikha Chandrasekaran, Michael G. Poulos, Michael Ginsberg, Daniel J. Nolan, Olivier Elemento, Brent L. Wood, Jennifer E. Adair, Shahin Rafii, Hans-Peter Kiem
Astrocytes are integral components of the homeostatic neural network as well as active participants in pathogenesis of and recovery from nearly all neurological conditions. Evolutionarily, compared with lower vertebrates and nonhuman primates, humans have an increased astrocyte-to-neuron ratio; however, a lack of effective models has hindered the study of the complex roles of human astrocytes in intact adult animals. Here, we demonstrated that after transplantation into the cervical spinal cords of adult mice with severe combined immunodeficiency (SCID), human pluripotent stem cell–derived (PSC-derived) neural progenitors migrate a long distance and differentiate to astrocytes that nearly replace their mouse counterparts over a 9-month period. The human PSC-derived astrocytes formed networks through their processes, encircled endogenous neurons, and extended end feet that wrapped around blood vessels without altering locomotion behaviors, suggesting structural, and potentially functional, integration into the adult mouse spinal cord. Furthermore, in SCID mice transplanted with neural progenitors derived from induced PSCs from patients with ALS, astrocytes were generated and distributed to a similar degree as that seen in mice transplanted with healthy progenitors; however, these mice exhibited motor deficit, highlighting functional integration of the human-derived astrocytes. Together, these results indicate that this chimeric animal model has potential for further investigating the roles of human astrocytes in disease pathogenesis and repair.
Hong Chen, Kun Qian, Wei Chen, Baoyang Hu, Lisle W. Blackbourn IV, Zhongwei Du, Lixiang Ma, Huisheng Liu, Karla M. Knobel, Melvin Ayala, Su-Chun Zhang
For a targeted cancer vaccine to be effective, the antigen of interest needs to be naturally processed and presented on MHC by the target cell or an antigen-presenting cell (APC) in the tumor stroma. The presence of these characteristics is often assumed based on animal models, evaluation of antigen-overexpressing APCs in vitro, or assays of material-consuming immune precipitation from fresh solid tissue. Here, we evaluated the use of an alternative approach that uses the proximity ligation assay (PLA) to identify the presentation of an MHC class II–restricted antigen in paraffin-embedded tissue sections from patients with brain tumors. This approach required a specific antibody directed against the epitope that was presented. We used an antibody that specifically binds an epitope of mutated isocitrate dehydrogenase type 1 (IDH1R132H), which is frequently expressed in gliomas and other types of tumors. In situ PLA showed that the IDH1R132H epitope colocalizes with MHC class II in IDH1R132H-mutated glioma tissue. Moreover, PLA demonstrated colocalization between the class II epitope-containing melanoma antigen New York esophageal 1 and MHC class II. Collectively, our data suggest that PLA may be a useful tool to acquire information on whether an antigen is presented in situ, and this technique has potential to guide clinical studies that use antigen-specific cancer immunotherapy.
Lukas Bunse, Theresa Schumacher, Felix Sahm, Stefan Pusch, Iris Oezen, Katharina Rauschenbach, Marina Gonzalez, Gergely Solecki, Matthias Osswald, David Capper, Benedikt Wiestler, Frank Winkler, Christel Herold-Mende, Andreas von Deimling, Wolfgang Wick, Michael Platten
The hypothalamus is the central regulator of systemic energy homeostasis, and its dysfunction can result in extreme body weight alterations. Insights into the complex cellular physiology of this region are critical to the understanding of obesity pathogenesis; however, human hypothalamic cells are largely inaccessible for direct study. Here, we developed a protocol for efficient generation of hypothalamic neurons from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) obtained from patients with monogenetic forms of obesity. Combined early activation of sonic hedgehog signaling followed by timed NOTCH inhibition in human ESCs/iPSCs resulted in efficient conversion into hypothalamic NKX2.1+ precursors. Application of a NOTCH inhibitor and brain-derived neurotrophic factor (BDNF) further directed the cells into arcuate nucleus hypothalamic-like neurons that express hypothalamic neuron markers proopiomelanocortin (POMC), neuropeptide Y (NPY), agouti-related peptide (AGRP), somatostatin, and dopamine. These hypothalamic-like neurons accounted for over 90% of differentiated cells and exhibited transcriptional profiles defined by a hypothalamic-specific gene expression signature that lacked pituitary markers. Importantly, these cells displayed hypothalamic neuron characteristics, including production and secretion of neuropeptides and increased p-AKT and p-STAT3 in response to insulin and leptin. Our results suggest that these hypothalamic-like neurons have potential for further investigation of the neurophysiology of body weight regulation and evaluation of therapeutic targets for obesity.
Liheng Wang, Kana Meece, Damian J. Williams, Kinyui Alice Lo, Matthew Zimmer, Garrett Heinrich, Jayne Martin Carli, Charles A. Leduc, Lei Sun, Lori M. Zeltser, Matthew Freeby, Robin Goland, Stephen H. Tsang, Sharon L. Wardlaw, Dieter Egli, Rudolph L. Leibel
The demonstrated ability to differentiate both human embryonic stem cells (hESCs) and patient-derived induced pluripotent stem cells (hiPSCs) into hepatocyte-like cells (HLCs) holds great promise for both regenerative medicine and liver disease research. Here, we determined that, despite an immature phenotype, differentiated HLCs are permissive to hepatitis C virus (HCV) infection and mount an interferon response to HCV infection in vitro. HLCs differentiated from hESCs and hiPSCs could be engrafted in the liver parenchyma of immune-deficient transgenic mice carrying the urokinase-type plasminogen activator gene driven by the major urinary protein promoter. The HLCs were maintained for more than 3 months in the livers of chimeric mice, in which they underwent further maturation and proliferation. These engrafted and expanded human HLCs were permissive to in vivo infection with HCV-positive sera and supported long-term infection of multiple HCV genotypes. Our study demonstrates efficient engraftment and in vivo HCV infection of human stem cell–derived hepatocytes and provides a model to study chronic HCV infection in patient-derived hepatocytes, action of antiviral therapies, and the biology of HCV infection.
Arnaud Carpentier, Abeba Tesfaye, Virginia Chu, Ila Nimgaonkar, Fang Zhang, Seung Bum Lee, Snorri S. Thorgeirsson, Stephen M. Feinstone, T. Jake Liang
Animal models suggest that acetylcarnitine production is essential for maintaining metabolic flexibility and insulin sensitivity. Because current methods to detect acetylcarnitine involve biopsy of the tissue of interest, noninvasive alternatives to measure acetylcarnitine concentrations could facilitate our understanding of its physiological relevance in humans. Here, we investigated the use of long–echo time (TE) proton magnetic resonance spectroscopy (1H-MRS) to measure skeletal muscle acetylcarnitine concentrations on a clinical 3T scanner. We applied long-TE 1H-MRS to measure acetylcarnitine in endurance-trained athletes, lean and obese sedentary subjects, and type 2 diabetes mellitus (T2DM) patients to cover a wide spectrum in insulin sensitivity. A long-TE 1H-MRS protocol was implemented for successful detection of skeletal muscle acetylcarnitine in these individuals. There were pronounced differences in insulin sensitivity, as measured by hyperinsulinemic-euglycemic clamp, and skeletal muscle mitochondrial function, as measured by phosphorus-MRS (31P-MRS), across groups. Insulin sensitivity and mitochondrial function were highest in trained athletes and lowest in T2DM patients. Skeletal muscle acetylcarnitine concentration showed a reciprocal distribution, with mean acetylcarnitine concentration correlating with mean insulin sensitivity in each group. These results demonstrate that measuring acetylcarnitine concentrations with 1H-MRS is feasible on clinical MR scanners and support the hypothesis that T2DM patients are characterized by a decreased formation of acetylcarnitine, possibly underlying decreased insulin sensitivity.
Lucas Lindeboom, Christine I. Nabuurs, Joris Hoeks, Bram Brouwers, Esther Phielix, M. Eline Kooi, Matthijs K.C. Hesselink, Joachim E. Wildberger, Robert D. Stevens, Timothy Koves, Deborah M. Muoio, Patrick Schrauwen, Vera B. Schrauwen-Hinderling
Millions of patients worldwide are affected by craniofacial deformations caused by congenital defects or trauma. Current surgical interventions have limited therapeutic outcomes; therefore, methods that would allow cartilage restoration are of great interest. A number of studies on embryonic limb development have shown that chondrogenesis is initiated by cellular condensation, during which mesenchymal progenitors aggregate and form 3D structures. Here, we demonstrated efficient regeneration of avascular elastic cartilage from in vitro–grown mesenchymal condensation, which recapitulated the early stages of chondrogenesis, including transient vascularization. After transplantation of vascularized condensed progenitors into immunodeficient mice, we used an intravital imaging approach to follow cartilage maturation. We determined that endothelial cells are present inside rudimentary cartilage (mesenchymal condensation) prior to cartilage maturation. Recreation of endothelial interactions in culture enabled a recently identified population of adult elastic cartilage progenitors to generate mesenchymal condensation in a self-driven manner, without requiring the support of exogenous inductive factors or scaffold materials. Moreover, the culture-grown 3D condensed adult–derived progenitors were amenable to storage via simple freezing methods and efficiently reconstructed 3D elastic cartilage upon transplantation. Together, our results indicate that transplantation of endothelialized and condensed progenitors represents a promising approach to realizing a regenerative medicine treatment for craniofacial deformations.
Takanori Takebe, Shinji Kobayashi, Hiromu Suzuki, Mitsuru Mizuno, Yu-Min Chang, Emi Yoshizawa, Masaki Kimura, Ayaka Hori, Jun Asano, Jiro Maegawa, Hideki Taniguchi
Muscle satellite cells promote regeneration and could potentially improve gene delivery for treating muscular dystrophies. Human satellite cells are scarce; therefore, clinical investigation has been limited. We obtained muscle fiber fragments from skeletal muscle biopsy specimens from adult donors aged 20 to 80 years. Fiber fragments were manually dissected, cultured, and evaluated for expression of myogenesis regulator PAX7. PAX7+ satellite cells were activated and proliferated efficiently in culture. Independent of donor age, as few as 2 to 4 PAX7+ satellite cells gave rise to several thousand myoblasts. Transplantation of human muscle fiber fragments into irradiated muscle of immunodeficient mice resulted in robust engraftment, muscle regeneration, and proper homing of human PAX7+ satellite cells to the stem cell niche. Further, we determined that subjecting the human muscle fiber fragments to hypothermic treatment successfully enriches the cultures for PAX7+ cells and improves the efficacy of the transplantation and muscle regeneration. Finally, we successfully altered gene expression in cultured human PAX7+ satellite cells with Sleeping Beauty transposon–mediated nonviral gene transfer, highlighting the potential of this system for use in gene therapy. Together, these results demonstrate the ability to culture and manipulate a rare population of human tissue-specific stem cells and suggest that these PAX7+ satellite cells have potential to restore gene function in muscular dystrophies.
Andreas Marg, Helena Escobar, Sina Gloy, Markus Kufeld, Joseph Zacher, Andreas Spuler, Carmen Birchmeier, Zsuzsanna Izsvák, Simone Spuler
The NF-κB signaling pathway is implicated in various inflammatory diseases, including rheumatoid arthritis (RA); therefore, inhibition of this pathway has the potential to ameliorate an array of inflammatory diseases. Given that NF-κB signaling is critical for many immune cell functions, systemic blockade of this pathway may lead to detrimental side effects. siRNAs coupled with a safe and effective delivery nanoplatform may afford the specificity lacking in systemic administration of small-molecule inhibitors. Here we demonstrated that a melittin-derived cationic amphipathic peptide combined with siRNA targeting the p65 subunit of NF-κB (p5RHH-p65) noncovalently self-assemble into stable nanocomplexes that home to the inflamed joints in a murine model of RA. Specifically, administration of p5RHH-p65 siRNA nanocomplexes abrogated inflammatory cytokine expression and cellular influx into the joints, protected against bone erosions, and preserved cartilage integrity. The p5RHH-p65 siRNA nanocomplexes potently suppressed early inflammatory arthritis without affecting p65 expression in off-target organs or eliciting a humoral response after serial injections. These data suggest that this self-assembling, largely nontoxic platform may have broad utility for the specific delivery of siRNA to target and limit inflammatory processes for the treatment of a variety of diseases.
Hui-fang Zhou, Huimin Yan, Hua Pan, Kirk K. Hou, Antonina Akk, Luke E. Springer, Ying Hu, J. Stacy Allen, Samuel A. Wickline, Christine T.N. Pham