News Article | March 18, 2016
An embryonic stem cell with just half a genome was generated by scientists, according to findings published in the journal Nature. The haploid stem cells are the first human cells capable of cell division with just one copy of the genome, allowing easier genetic manipulation and analysis, according to the American and Israeli researchers. “One of the greatest advantages of using haploid human cells is that it is much easier to edit their genes,” said Ido Sagi, the also of the Hebrew University of Jerusalem, who corresponded briefly with Laboratory Equipment by email. The technique involved triggering unfertilized human egg cells, highlighting the genome with a fluorescent dye and then isolating the haploid stem cells, according to the scientists, some of whom are from Columbia University Medical Center and the New York Stem Cell Foundation Research Institute. The stem cells are pluripotent, with the ability to become a litany of other kinds of cells, including those of major organs. “This study has given us a new type of human stem cell which will have an important impact on human genetic and medical research,” said Nissim Benvenisty, director of the Azrieli Center for Stem Cells and Genetic Research at the Hebrew University of Jerusalem. “These cells will provide researchers with a novel tool for improving our understanding of human development.” Sagi told Laboratory Equipment that the easiest application of the technique would be for genetic screening. The haploid cells were easier to scan for a particular mutation which allowed resistance to certain cellular toxicity – and the analysis could translate to all sorts of genetic variants, he said. “Unlike many previous genetic studies in humans, which focused on editing specific genes, with haploid cells we are not limited to predetermined, targeted genetic changes,” said Sagi. “This means that we can generate millions of different mutations, forming a library of mutants that can help us understand what genes in the genome are involved in specific biological processes that interest us.” Because the 23-chromosome stem cells are easier to work with than the natural diploid cells humans developed through evolution, it could provide a new look into the fundamentals of life, they add. “We expect that haploid human embryonic stem cells will provide novel means for studying human functional genomics and development,” the authors conclude. Previous breakthroughs have produced haploid stem cells from fish and mice - which have proven valuable in genetic analysis, according to the scientific literature.
News Article | December 12, 2016
NEW YORK NY (December 12, 2016)--Columbia University Medical Center (CUMC) researchers have discovered that a deficiency of the enzyme prohormone covertase (PC1) in the brain is linked to most of the neuro-hormonal abnormalities in Prader-Willi syndrome, a genetic condition that causes extreme hunger and severe obesity beginning in childhood. The discovery provides insight into the molecular mechanisms underlying the syndrome and highlights a novel target for drug therapy. The findings were published online today in the Journal of Clinical Investigation. "While we've known for some time which genes are implicated in Prader-Willi syndrome, it has not been clear how those mutations actually trigger the disease," said lead author Lisa C. Burnett, PhD, a post-doctoral research scientist in pediatrics at CUMC. "Now that we have found a key link between these mutations and the syndrome's major hormonal features, we can begin to search for new, more precisely targeted therapies." An estimated one in 15,000 people have Prader-Willi syndrome (PWS). The syndrome is caused by abnormalities in a small region of chromosome 15, which leads to dysfunction in the hypothalamus--which contains cells that regulate hunger and satiety--and other regions of the brain. A defining characteristic of PWS is insatiable hunger. People with PWS typically have extreme obesity, reduced growth hormone and insulin levels, excessive levels of ghrelin (a hormone that triggers hunger), and developmental disabilities. There is no cure and few effective treatments for PWS. Dr. Burnett and her colleagues used stem cell techniques to convert skin cells from PWS patients and unaffected controls into brain cells. Analysis of the stem cell-derived neurons revealed significantly reduced levels of PC1 in the patients' cells, compared to the controls. The cells from PWS patients also had abnormally low levels of a protein, NHLH2, which is made by NHLH2, a gene that also helps to produce PC1. To confirm whether PC1 deficiency plays a role in PWS, the researchers examined transgenic mice that do not express Snord116, a gene that is deleted in the region of chromosome 15 that is associated with PWS. The mice were found to be deficient in NHLH2 and PC1 and displayed most of the hormone-related abnormalities seen in PWS, according to study leader Rudolph L. Leibel, MD, professor of pediatrics and medicine and co-director of the Naomi Berrie Diabetes Center at CUMC. "The findings strongly suggest that PC1 is a good therapeutic target for PWS," said Dr. Burnett. "There doesn't seem to be anything wrong with the gene that makes PC1--it's just not getting activated properly. If we could elevate levels of PC1 using drugs, we might be able to alleviate some of the symptoms of the syndrome." "This is an outstanding example how research on human stem cells can lead to novel insight into a disease and provide a platform for the testing of new therapies," said Dieter Egli, PhD, a stem cell scientist who is an assistant professor of developmental cell biology (in Pediatrics) and a senior author on the paper. "This study changes how we think about this devastating disorder," said Theresa Strong, PhD, chair of the scientific advisory board of the Foundation for Prader-Willi Research and the mother of a child with PWS. "The symptoms of PWS have been very confusing and hard to reconcile. Now that we have an explanation for the wide array of symptoms, we can move forward with developing a drug that addresses their underlying cause, instead of treating each symptom individually." Following the findings reported in this paper, the Columbia research team began collaborating with Levo Therapeutics, a PWS-focused biotechnology company, to translate the current research into therapeutics. The study is titled, "Deficiency in prohormone convertase PC1 impairs prohormone processing in Prader-Willi syndrome." The other contributors are: Charles A. LeDuc (CUMC), Carlos R. Sulsona (University of Florida College of Medicine Gainesville, FL), Daniel Paull (New York Stem Cell Foundation Research Institute, New York, NY), Richard Rausch (CUMC), Sanaa Eddiry (Université Paul Sabatier, Toulouse, France), Jayne F. Martin Carli (CUMC), Michael V. Morabito (CUMC), Alicja A. Skowronski (CUMC), Gabriela Hubner (Packer Collegiate Institute), Matthew Zimmer (New York Stem Cell Foundation Research Institute), Liheng Wang (CUMC), Robert Day (Université de Sherbrooke, Quebec, Canada), Brynn Levy (CUMC), Ilene Fennoy (CUMC), Beatrice Dubern (Sorbonne University, University Pierre et Marie-Curie, Paris, France), Christine Poitou (Sorbonne University), Karine Clement (Sorbonne University), Merlin G. Butler (Kansas University Medical Center, Kansas City, KS), Michael Rosenbaum (CUMC), Jean Pierre Salles (Université de Toulouse. Toulouse, France), Maithe Tauber (Université de Toulouse), Daniel J. Driscoll (University of Florida College of Medicine), and Dieter Egli (CUMC and New York Stem Cell Foundation Research Institute). The study was supported by grants from the Foundation for Prader-Willi Research, Russell Berrie Foundation, Rudin Foundation, The New York Stem Cell Foundation, Helmsley Foundation, and National Institutes of Health (RO1DK52431 and P30 DK26687). The authors declare no conflicts of interest. Columbia University Medical Center provides international leadership in basic, preclinical, and clinical research; medical and health sciences education; and patient care. The medical center trains future leaders and includes the dedicated work of many physicians, scientists, public health professionals, dentists, and nurses at the College of Physicians and Surgeons, the Mailman School of Public Health, the College of Dental Medicine, the School of Nursing, the biomedical departments of the Graduate School of Arts and Sciences, and allied research centers and institutions. Columbia University Medical Center is home to the largest medical research enterprise in New York City and State and one of the largest faculty medical practices in the Northeast. The campus that Columbia University Medical Center shares with its hospital partner, NewYork-Presbyterian, is now called the Columbia University Irving Medical Center. For more information, visit cumc.columbia.edu or columbiadoctors.org.
Yeh C.,Cornell University |
Li A.,Cornell University |
Li A.,New York Stem Cell Foundation Research Institute |
Chuang J.-Z.,Cornell University |
And 4 more authors.
Developmental Cell | Year: 2013
Primary cilia undergo cell-cycle-dependent assembly and disassembly. Emerging data suggest that ciliary resorption is a checkpoint for S phase reentry and that the activation of phospho(T94)Tctex-1 couples these two events. However, the environmental cues and molecular mechanisms that trigger these processes remain unknown. Here, we show that insulin-like growth-1 (IGF-1) accelerates G1-S progression by causing cilia to resorb. The mitogenic signals of IGF-1 are predominantly transduced through IGF-1 receptor (IGF-1R) on the cilia of fibroblasts and epithelial cells. At the base of the cilium, phosphorylated IGF-1R activates an AGS3-regulated Gβγ signaling pathway that subsequently recruits phospho(T94)Tctex-1 to the transition zone. Perturbing any component of this pathway in cortical progenitors induces premature neuronal differentiation at the expense of proliferation. These data suggest that during corticogenesis, a cilium-transduced, noncanonical IGF-1R-Gβγ-phospho(T94)Tctex-1 signaling pathway promotes the proliferation of neural progenitors through modulation of ciliary resorption and G1 length. © 2013 Elsevier Inc.
Johannesson B.,New York Stem Cell Foundation Research Institute |
Sui L.,Columbia University |
Freytes D.O.,New York Stem Cell Foundation Research Institute |
Creusot R.J.,Columbia University |
And 2 more authors.
EMBO Journal | Year: 2015
The discovery of insulin more than 90 years ago introduced a life-saving treatment for patients with type 1 diabetes, and since then, significant progress has been made in clinical care for all forms of diabetes. However, no method of insulin delivery matches the ability of the human pancreas to reliably and automatically maintain glucose levels within a tight range. Transplantation of human islets or of an intact pancreas can in principle cure diabetes, but this approach is generally reserved for cases with simultaneous transplantation of a kidney, where immunosuppression is already a requirement. Recent advances in cell reprogramming and beta cell differentiation now allow the generation of personalized stem cells, providing an unlimited source of beta cells for research and for developing autologous cell therapies. In this review, we will discuss the utility of stem cell-derived beta cells to investigate the mechanisms of beta cell failure in diabetes, and the challenges to develop beta cell replacement therapies. These challenges include appropriate quality controls of the cells being used, the ability to generate beta cell grafts of stable cellular composition, and in the case of type 1 diabetes, protecting implanted cells from autoimmune destruction without compromising other aspects of the immune system or the functionality of the graft. Such novel treatments will need to match or exceed the relative safety and efficacy of available care for diabetes. Dieter Egli & colleagues provide a stem cell perspective on pancreatic beta-cells for diabetes therapies and disease modeling. © 2015 The Authors.
Douvaras P.,New York Stem Cell Foundation Research Institute |
Fossati V.,New York Stem Cell Foundation Research Institute
Nature Protocols | Year: 2015
In the CNS, oligodendrocytes act as the myelinating cells. Oligodendrocytes have been identified to be key players in several neurodegenerative disorders. This protocol describes a robust, fast and reproducible differentiation protocol to generate human oligodendrocytes from pluripotent stem cells (PSCs) using a chemically defined, growth factor-rich medium. Within 8 d, PSCs differentiate into paired box 6-positive (PAX6 +) neural stem cells, which give rise to OLIG2 + progenitors by day 12. Oligodendrocyte lineage transcription factor 2-positive (OLIG2 +) cells begin to express the transcription factor NKX2.2 around day 18, followed by SRY-box 10 (SOX10) around day 40. Oligodendrocyte progenitor cells (OPCs) that are positive for the cell surface antigen recognized by the O4 antibody (O4 +) appear around day 50 and reach, on average, 43% of the cell population after 75 d of differentiation. O4 + OPCs can be isolated by cell sorting for myelination studies, or they can be terminally differentiated to myelin basic protein-positive (MBP +) oligodendrocytes. This protocol also describes an alternative strategy for markedly reducing the length and the costs of the differentiation and generating a 1/430% O4 + cells after only 55 d of culture. © 2015 Nature America, Inc. All rights reserved.
Sproul A.A.,New York Stem Cell Foundation Research Institute |
Sproul A.A.,Columbia University
Molecular Aspects of Medicine | Year: 2015
Human pluripotent stem cells (PSCs) have the capacity to revolutionize medicine by allowing the generation of functional cell types such as neurons for cell replacement therapy. However, the more immediate impact of PSCs on treatment of Alzheimer's disease (AD) will be through improved human AD model systems for mechanistic studies and therapeutic screening. This review will first briefly discuss different types of PSCs and genome-editing techniques that can be used to modify PSCs for disease modeling or for personalized medicine. This will be followed by a more in depth analysis of current AD iPSC models and a discussion of the need for more complex multicellular models, including cell types such as microglia. It will finish with a discussion on current clinical trials using PSC-derived cells and the long-term potential of such strategies for treating AD. © 2015 Elsevier Ltd. All rights reserved.
De Peppo G.M.,New York Stem Cell Foundation Research Institute |
Marolt D.,New York Stem Cell Foundation Research Institute
Expert Opinion on Biological Therapy | Year: 2014
Recent developments in nuclear reprogramming allow the generation of patient-matched stem cells with broad potential for applications in cell therapies, disease modeling and drug discovery. An increasing body of work is reporting the derivation of lineage-specific progenitors from human-induced pluripotent stem cells (hiPSCs), which could in the near future be used to engineer personalized tissue substitutes, including those for reconstructive therapies of bone. Although the potential clinical impact of such technology is not arguable, significant challenges remain to be addressed before hiPSC-derived progenitors can be employed to engineer bone substitutes of clinical relevance. The most important challenge is indeed the construction of personalized multicellular bone substitutes for the treatment of complex skeletal defects that integrate fast, are immune tolerated and display biofunctionality and long-term safety. As recent studies suggest, the merging of iPSC technology with advanced biomaterials and bioreactor technologies offers a way to generate bone substitutes in a controllable, automated manner with potential to meet the needs for scale-up and requirements for translation into clinical practice. It is only via the use of state-of-the-art cell culture technologies, process automation under GMP-compliant conditions, application of appropriate engineering strategies and compliance with regulatory policies that personalized lab-made bone grafts can start being used to treat human patients. © 2014 Informa UK, Ltd.
De Peppo G.M.,New York Stem Cell Foundation Research Institute |
Vunjak-Novakovic G.,Columbia University |
Marolt D.,New York Stem Cell Foundation Research Institute
Methods in Molecular Biology | Year: 2014
Human pluripotent stem cells represent an unlimited source of skeletal tissue progenitors for studies of bone biology, pathogenesis, and the development of new approaches for bone reconstruction and therapies. In order to construct in vitro models of bone tissue development and to grow functional, clinical-size bone substitutes for transplantation, cell cultivation in three-dimensional environments composed of porous osteoconductive scaffolds and dynamic culture systems-bioreactors-has been studied. Here, we describe a stepwise procedure for the induction of human embryonic and induced pluripotent stem cells (collectively termed PSCs) into mesenchymal-like progenitors, and their subsequent cultivation on decellularized bovine bone scaffolds in perfusion bioreactors, to support the development of viable, stable bone-like tissue in defined geometries. © 2014 Springer Science+Business Media New York.
Paquet D.,Rockefeller University |
Kwart D.,Rockefeller University |
Chen A.,Rockefeller University |
Sproul A.,New York Stem Cell Foundation Research Institute |
And 7 more authors.
Nature | Year: 2016
The bacterial CRISPR/Cas9 system allows sequence-specific gene editing in many organisms and holds promise as a tool to generate models of human diseases, for example, in human pluripotent stem cells. CRISPR/Cas9 introduces targeted double-stranded breaks (DSBs) with high efficiency, which are typically repaired by non-homologous end-joining (NHEJ) resulting in nonspecific insertions, deletions or other mutations (indels). DSBs may also be repaired by homology-directed repair (HDR) using a DNA repair template, such as an introduced single-stranded oligo DNA nucleotide (ssODN), allowing knock-in of specific mutations. Although CRISPR/Cas9 is used extensively to engineer gene knockouts through NHEJ, editing by HDR remains inefficient and can be corrupted by additional indels, preventing its widespread use for modelling genetic disorders through introducing disease-associated mutations. Furthermore, targeted mutational knock-in at single alleles to model diseases caused by heterozygous mutations has not been reported. Here we describe a CRISPR/Cas9-based genome-editing framework that allows selective introduction of mono- and bi-allelic sequence changes with high efficiency and accuracy. We show that HDR accuracy is increased dramatically by incorporating silent CRISPR/Cas-blocking mutations along with pathogenic mutations, and establish a method termed ' CORRECT' for scarless genome editing. By characterizing and exploiting a stereotyped inverse relationship between a mutation's incorporation rate and its distance to the DSB, we achieve predictable control of zygosity. Homozygous introduction requires a guide RNA targeting close to the intended mutation, whereas heterozygous introduction can be accomplished by distance-dependent suboptimal mutation incorporation or by use of mixed repair templates. Using this approach, we generated human induced pluripotent stem cells with heterozygous and homozygous dominant early onset Alzheimer's disease-causing mutations in amyloid precursor protein (APP Swe) and presenilin 1 (PSEN1 M146V) and derived cortical neurons, which displayed genotype-dependent disease-associated phenotypes. Our findings enable efficient introduction of specific sequence changes with CRISPR/Cas9, facilitating study of human disease. © 2016 Macmillan Publishers Limited. All rights reserved.
Zhang Y.S.,Columbia University |
Sevilla A.,Mount Sinai School of Medicine |
Sevilla A.,New York Stem Cell Foundation Research Institute |
Wan L.Q.,Columbia University |
And 3 more authors.
Stem Cells | Year: 2013
Developmental gradients of morphogens and the formation of boundaries guide the choices between self-renewal and differentiation in stem cells. Still, surprisingly little is known about gene expression signatures of differentiating stem cells at the boundaries between regions. We thus combined inducible gene expression with a microfluidic technology to pattern gene expression in murine embryonic stem cells. Regional depletion of the Nanog transcriptional regulator was achieved through the exposure of cells to microfluidic gradients of morphogens. In this way, we established pluripotency-differentiation boundaries between Nanog expressing cells (pluripotency zone) and Nanog suppressed cells (early differentiation zone) within the same cell population, with a gradient of Nanog expression across the individual cell colonies, to serve as a mimic of the developmental process. Using this system, we identified strong interactions between Nanog and its target genes by constructing a network with Nanog as the root and the measured levels of gene expression in each region. Gene expression patterns at the pluripotency-differentiation boundaries recreated in vitro were similar to those in the developing blastocyst. This approach to the study of cellular commitment at the boundaries between gene expression domains, a phenomenon critical for understanding of early development, has potential to benefit fundamental research of stem cells and their application in regenerative medicine. Stem Cells 2013;31:1806-1815 © AlphaMed Press.