Chair, Program Committee: Hynek Wichterle hw350@columbia.edu
General inquiries: Dola Sengupta ds2865@columbia.edu
Columbia Stem Cell Day 2012
May 7, 2012
Myrna Daniels Auditorium and the Riverview Terrace; Vivian and Seymour
Family Heart Center; 173 Fort Washington Avenue, New York, NY 10032
P
ROGRAM
3
Columbia Stem Cell Day
May 7, 2012
9:30 AM
Breakfast
10:00 AM
Opening remarks
Lee Goldman,
Executive Vice President for Health & Biomedical Sciences
Dean of the Faculties of Health Sciences & of Medicine
10:05 AM
Studies of hematopoietic stem cell transplantation
Megan Sykes
10:25 AM
Dclk1 labels quiescent pancreatic progenitor and cancer initiating cells
Christoph B. Westphalen (lab of Timothy Wang)
10:40 AM
Intestinal Stem Cell Maintenance in the Adult Drosophila Posterior Midgut
Ben Ohlstein
11:00 AM
Coffee break
11:20 AM
Synovium-derived stem cells for cartilage tissue engineering
Clark Hung
11:40 AM
Mapping biochemical pathways underlying the pathology of Giant Axonal Neuropathy using iPS cells
Bethany L. Johnson-Kerner (Project A.L.S. lab, Henderson & Wichterle)
11:55 AM
Generation of lung and airway epithelial cells from human pluripotent stem cells
Hans Snoeck
12:15 PM
LUNCH/Poster session
2:00 PM
Stem cells in the adrenal cortex
Ed Laufer
2:20 PM
Spatial and temporal regulation of signaling pathways in hESCs using microfluidic technologies
Elisa Cimetta (lab of Gordana Vunjak-Novakovic)
2:35 PM
Regulating Islet Cell Lineages
Lori Sussel
2:55 PM
Coffee break
3:25 PM
Directed differentiation and transdifferentiation to prostate epithelia
Flaminia Talos (lab of Michael Shen)
3:40 PM
Turning skin into neurons - a new perspective on an old problem
Oliver Hobert
4
Columbia Stem
Cell Day
Short talks
Dclk1 labels quiescent pancreatic progenitor and
cancer initiating cells
Christoph B. Westphalen, Michael Quante, Daniel Worthley,
Samuel Asfaha, Helen Remotti, Kenneth P. Olive, Timothy C.
Wang
Introduction: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death in the US. Despite major efforts pancreatic cancer has the highest mortality rate of all major cancers with a 5-year survival rate below 5% while the survival rate for the disease has not improved substantially in nearly 40 years. Recently, doublecortin-like kinase 1 protein (Dclk1) was proposed as a marker of putative quiescent stem cells in the gastrointestinal tract and in the pancreas although no genetic fate mapping was performed. Additionally, it was demonstrated that Dclk1 cells expand in the setting of inflammation, hyperplasia and metaplasia. While clinical relevant mouse models of PDAC exist the identity of the cell of origin for pancreatic cancer is still debated.
Aim: In order to investigate the role of Dclk1 cells in pancreatic health and disease we generated a BAC transgenic mouse line (Dclk1-CreERT) that expresses tamoxifen-inducible Cre recombinase under the control of the endogenous Dclk1 gene regulation.
Results: Using genetic lineage tracing, we demonstrate that Dclk1 labels rare long-lived, quiescent progenitor cells in the pancreas that can give rise to ductal and acinar tissue. Furthermore, Dclk1 cells were found to expand in the setting of pancreatic inflammation and malignancy. Importantly, the Dclk1 lineage contributed significantly to pancreatic regeneration after caerulein-induced pancreatitis. Finally, expression of mutant K-Ras (G12D) in Dclk-1 cells leads to rapid onset (2-3 months) of invasive pancreatic ductal adenocarcinoma that could be further accelerated by caerulein-induced pancreatic injury (1-2 months).
Conclusion: Dclk1 labels rare quiescent progenitor cells in the pancreas. These cells take part in the regeneration of the gland after injury and are capable of giving rise to pancreatic cancer. Therefore, we propose that Dclk1 could label the cell of origin in pancreatic cancer.
Mapping biochemical pathways underlying the
pathology of Giant Axonal Neuropathy using iPS
cells
Bethany L. Johnson-Kerner, Faizzan S. Ahmad, Alejandro Garcia
Diaz, J. Palmer Greene, Wendy K. Chung, Steven J. Gray, R. Jude
Samulski, Scott Noggle, Christopher E. Henderson, Hynek
Wichterle*
Giant axonal neuropathy (GAN) is caused by autosomal recessive mutations in GAN, which encodes gigaxonin, a ubiquitously expressed cytoplasmic protein. Patients with GAN present in their first decade with loss of motor and sensory function, but the cellular functions of gigaxonin remain unknown. Currently there is no treatment for GAN, and the life expectancy is typically less than thirty years. While several mouse models of GAN have been created, none demonstrate the early phenotypic onset and severe motor dysfunction displayed by the patients, suggesting that gigaxonin may have human-specific functions. Here we report the derivation of GAN induced pluripotent stem cells (iPSCs) from three patients with different combinations of GAN mutations. Motor neurons (MNs) differentiated from GAN iPSCs recapitulate the key GAN pathophysiological phenotype found in patients: accumulation of neuronal intermediate filaments (nIFs) at an early time point. High nIF levels can be reversed by replacing gigaxonin with a viral vector, suggesting that they are
gigaxonin-dependent, but the link between gigaxonin and nIFs continued to be unknown. To determine that link, we sought to identify the binding partners of gigaxonin using a combination of affinity purification and mass spectrometry in HEK293 cells. Notably, seven intermediate filament proteins were pulled-down with gigaxonin, including vimentin which aggregates in patient fibroblasts and peripherin which aggregates in GAN iPS-MNs. The interaction between gigaxonin and vimentin was verified by independent co-immunoprecipitation experiments, and provides the first link between the loss of gigaxonin and the disease phenotype in GAN patients. Studies are ongoing to define a possible consensus binding site for the regulation of IFs by gigaxonin. Understanding the human-specific roles of gigaxonin in the maintenance of the cytoskeletal network in motor neurons should provide direction for future therapeutic initiatives.
Spatial and temporal regulation of signaling
pathways in hESCs using microfluidic technologies
E Cimetta, D Sirabella, K Yeager, G Vunjak-Novakovic
During development and regeneration, tissues emerge from coordinated sequences of stem cell renewal, specialization and assembly orchestrated by cascades of regulatory signals. While this complex in vivo milieu is poorly replicable using standard in vitro techniques, microscale technologies offer potential for conducting more sophisticated experiments at biologically relevant scales and with real-time insights into cellular responses.
We developed a microbioreactor providing complex sequences of time- and space-resolved concentration gradients in 3D cell culture settings, and coupling application of fast dynamic changes of environmental signals with versatile, high-throughput operations and imaging compatibility.
Our device comprises a matrix of conical microwells accommodating 3D cell constructs which are protected from potentially harmful shear forces and exposed to stable concentration gradients generated by an integrated microfluidic platform. Mathematical modeling of flow and mass transport is used to determine the shape of the gradients and to predict the fast dynamic concentration changes of characteristic molecular species. The cell culture area in the assembled platform is optically transparent, allowing on line observations using standard microscopes.
H1-BAR-Ve us hE“Cs dri i g the W t/β-catenin–dependent expression of Venus, hES02 and iPSc were exposed to time- and space-resolved concentration gradients of multiple factors. Wnt3a, ActivinA, and BMP4 were chosen for being the main players in early stage fate specification and lineage commitment in the developing embryo.
Results will be presented correlating the behavior of differentiating Embryoid Bodies to their exposure to complex time- and space-resolved concentration gradients of mesodermal-inducing morphogens. We hypothesize that the application of complex regulatory patterns combining spatial and temporal gradients of molecular and physical factors to hESC and iPSc cultures would provide predictable in vitro models of development and disease.
Directed differentiation and transdifferentiation to
prostate epithelia
Flaminia Talos, Michael Shen
5
Posters
Heart Center Riverview Terrace
1
Biomarkers for growth factor priming in cartilage
tissue engineering using mass spectrometry and
ion mobility spectrometry
Elena Alegre-Aguarón (1), Sonal R. Sampat (1), J. Chloë Bulinski
(2), James L. Cook (3), Ryan M. Colligan (2), Lewis M. Brown (2),
Clark T. Hung (1)
(1) Department of Biomedical Engineering, Columbia University, New
York, NY (2) Department of Biological Sciences, Columbia University,
New York, NY (3) Comparative Orthopaedic Laboratory, University of
Missouri, Columbia, MO
Adult articular cartilage has a poor healing capacity, which leads to intense research on development of cell-based therapies for cartilage repair. The destruction of articular cartilage results in osteoarthritis (OA), which affects about 27 million Americans. To create functional tissue we need to mimic the native physiological environment of the tissue, optimizing expansion protocols. Cell passaging and priming with chemical or physical factors are often necessary steps in cell-based strategies for regenerative medicine. The ability to identify biomarkers that can act as predictors of cells with high capacity to form functional engineered cartilage will permit optimization of protocols for cartilage tissue engineering using various cell sources. Recent investigations have shown that chondrocytes and synovium-derived stem cells (SDSCs) are both promising cell sources for cartilage repair. The aim of this study was to investigate the impact of growth factor priming in 2D canine chondrocyte and SDSC cultures, through the identification of biomarkers, which can correlate to functional tissue elaboration in 3D. Label-free shotgun proteomics based on mass spectrometry was used. Hardware included a NanoAcquity liquid chromatograh and a Synapt G2 HDMS mass spectrometer enabled with both quadrupole and time-of-flight analyzers and an ion mobility spectrometer (IMS). IMS provided separation on the basis of cross-sectional area in addition to the conventional mass-to-charge ratio. This provided 2-dimensional peptide ion separation in the gas phase and substantially increased proteome depth of coverage. Spectra were analyzed with a massively parallel 448-core graphics processing unit workstation with 1 Teraflop processing power. Data mining and statistical analysis were performed with the Elucidator software.
Cartilage and synovium from adult canine knee joints (euthanized for other purposes) were digested using collagenase. Chondrocytes and synoviocytes were isolated and primed towards a chondrogenic lineage in 2D culture with a cocktail of growth factors (1 ng/mL TGF-β , g/ L FGF-2 and 10 ng/mL
PDGF-ββ; pri ed group . Cells e pa ded ithout the o ktail of growth factors
ser ed as the o trol u pri ed group . Protei s ere e tra ted at P for
primed and unprimed chondrocytes and SDSCs. A total of 3,684 proteins (represented by one or more peptides) were detected. Overall, the priming effect was greater for SDSCs than for chondrocytes. For chondrocytes, collagens (type I, II and V), plasminogen activator inhibitor-1 (suggesting collagen remodeling) and thrombospondin 1 (marker of articular cartilage) were downregulated and endothelial protein C receptor (stem cell marker) was upregulated. For SDSCs, a typical stem cell marker, aminopeptidase N (CD13), was 4.5-fold upregulated. Important transcription factors were differentially expressed. These transcription factors were associated with cell migration (pirin; upregulated), collagen biosynthesis (Y-box transcription factor; downregulated) and cytoskeleton (leucine-rich PPR motif-containing protein; upregulated). Other components of the extracellular matrix of cartilage such as biglycan and lumican were downregulated and some enzymes (C1 tetrahydrofolate synthase, D-3-phosphoglycerate dehydrogenase) involved in collagen synthesis were upregulated.
In this study, we demonstrated that growth factor priming effects were more pronounced in SDSCs compared to chondrocytes. SDSCs remained undifferentiated, while chondrocytes appeared to dedifferentiate, judging from stem cell marker expression and the downregulation of some collagens.
2
Accelerated
high-yield
generation
of
limb-innervating motor neurons from human stem cells
using small molecules
Mackenzie W. Amoroso
1,2, Gist F. Croft
1,2, Anne R. Davis
3,
Christopher E. Henderson
1,2, Hynek Wichterle
1,21
Project A.L.S./Jenifer Estess Laboratory for Stem Cell Research, New
York, NY 10032, USA
2
Departments of Pathology, Neurology and Neuroscience, Center for
Motor Neuron Biology and Disease (MNC), and Columbia Stem Cell
Initiative (CSCI), Columbia University, New York, NY 10032, USA
3
Department of Obstetrics and Gynecology, Columbia University
Medical Center, New York, NY 10032, USA
Human pluripotent stem cells are a promising source of diverse cells for developmental studies, cell transplantation, disease modeling and drug testing. However, their widespread use, even for intensely studied cell types like spinal motor neurons, is hindered by the long duration and low yields of existing protocols for in vitro differentiation and by the molecular heterogeneity of the populations generated. We report here a combination of small molecules that induce up to 50% motor neurons within 3 weeks from human pluripotent stem cells with defined subtype identities that are relevant to neurodegenerative diseases. Despite their accelerated differentiation, motor neuron subsets thus derived expressed combinations of HB9 and ISL1 and the columnar specific markers that mirror those observed in vivo in human fetal spinal cord. They also showed spontaneous and induced activity, and projected axons when grafted into developing chicken spinal cord. Strikingly, this novel protocol preferentially generates limb-innervating lateral motor column (FOXP1+) motor neurons, which are the most affected in amyotrophic lateral sclerosis (ALS). Access to high yield cultures of human limb-innervating motor neuron subtypes will facilitate in depth study of motor neuron subtype specific properties, disease modeling, and development of large-scale cell-based screening assays. Supported by Project A.L.S., P2ALS, NYSTEM contract #C024415, NIH GO grant 5 RC2 NS069395-02
3
Defining the fate of ghrelin-expressing cells in
pancreas
Luis Arnes
1, Jonathon Hill
1, Mark Magnuson
2and Lori Sussel
11
Dept. of Genetics and Development, Columbia University, New York,
NY 10032;
2Department of Cell and Developmental Biology,
Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
The ghrelin-produ i g ε ells ha e ee ide tified as the fifth e do ri e elltype within the islet of Langerhans. Ghrelin is expressed throughout the embryonic pancreas but its expression decreases postnatally, being barely detected in the adult mouse. Several knockout models where pancreas development is affected show that ghrelin cells can replace insulin-, glucagon- and PP- producing cells in a mutant islet. In this study, we have performed
li eage tra i g a al sis of the ε ells i ildt pe, Ngn3 and Nkx2.2 KO mouse models.
6
I su ar , our data li ks the o toge of PP ells to ε ells. Moreo er our
findings suggest that a population of ghrelin-expressing pancreatic progenitor cells have the potential to differentiate into all three pancreatic lineages. This project is funded by the BCBC grant #U01 DK089523
4
Intestinal Isthmus Keratin-19 Positive Stem Cells
Contribute to Intestinal Tumors
Samuel Asfaha, Christoph Westphalen, Russell Ericksen,
Johannes von Burstin, Sarah Stokes, Teresa Mastracci, Michael
Quante, Wataru Shibata, Daniel L. Worthley, Anil K. Rustgi,
Timothy C. Wang
Tumors are postulated to arise from tissue stem or progenitor cells. Previous studies have suggested a polyclonal origin for small intestinal tumors. In the intestine, keratin 19 (K19) is thought to mark a potential progenitor cell within the intestinal isthmus. We generated a new K19-BAC-CreER transgenic line to study the lineage of K19+ cells and determine whether K19+ might mark both normal and cancer initiating intestinal progenitors. K19-BAC-CreER mice were crossed to a ROSA26r(LacZ or GFP) reporter line. The offspring were induced with tamoxifen and studied in a AOM/DSS model of inflammatory colorectal carcinogenesis. In separate experiments, K19CreER/ROSA26r mice were crossed to ApcMin mice to examine the contribution of the K19 lineage to a genetic model of intestinal carcinogenesis. Tamoxifen-induced X-gal labeling occurred in ~20-50% of colonic and intestinal glands. The labeling persisted for greater than 52 weeks, consistent with K19 marking long-lived intestinal stem cells. In K19CreER/ROSA26r mice treated with AOM or DSS alone there was a dramatic increase in the labeling of contiguous X-gal positive glands, consistent with K19+ stem cell expansion and crypt fission. To test whether expansion of colonic progenitors represents a critical event in the initiation of tumors, we compared the pattern of lineage tracing in tumors relative to the timing of recombinase induction. When K19+ progenitor cells were labeled by tamoxifen prior to AOM and DSS, the majority of lineage traced tumors were entirely X-gal positive, suggesting they were each derived from a single recombined K19+ cell. Interestingly, when K19+ cells were labeled by tamoxifen after tumor initiation with AOM, tumors originated from both K19-Cre recombined and non-recombined cells, suggesting that AOM stimulates the K19+ progenitor cell to give rise to multiple K19+ daughter cells that contribute to the tumor. In contrast, in ApcMin mice, all X-gal marked small intestinal adenomas were derived from both K19 recombined and non-recombined cells. In summary, K19 marks intestinal and colonic stem cells located in the isthmus (above the crypt base), and these K19+ cells serve as cancer initiating cells in both carcinogen and genetic models of intestinal tumorigenesis. AOM-induced division of a K19+ cell, evidenced by contiguous crypt labeling and suggestive of symmetric cell division, appears to be an initiating event. Colonic tumors are polyclonal, but the multiple cancer initiating cells appear to arise from a single K19+ progenitor. The polyclonal adenomas from K19+ cells in APCmin mice, however, suggests much earlier cancer initiation, consistent with an inherited abnormality in stem cell division. We identify a key role for early stem cell amplification in cancer initiation, and a potentially important distinction between inherited and sporadic forms of intestinal cancer.
5
Yes-1 as a master regulator of the transition
between long-term to short-term hematopoietic
stem cells
Florence Borot, Michael Churchill, Peter Van Galen, John Dick
and Siddhartha Mukherjee
ABSTRACT: Hematopoiesis (the genesis of blood) originates in a population of long-term stem cells (LT-HSCs). LT-HSCs are typically deeply quiescent, and they make a crucial transition to short-term HSCs (ST-HSCs), which are more proliferative and give rise to the progenitor cells that eventually form the lineages of blood. The switch between quiescence & proliferation represents a crucial step in blood formation. Quiescence-modulating genes in HSCs could be modulated to increase stem cell transplantation efficacy or as a therapeutic strategy against AML. However, pathways specifying quiescence in LT-HSCs
remain very poorly characterized.
We performed an innovative, high throughput RNA interference screen to identify novel genes that might modulate HSC quiescence. A notable feature of this screen is that the screening is not performed on stem cells in isolation, but in the context of their ph siologi al i roe iro e t. We ter this i he
-ased s ree i g NB“ . This s ree ielded a ki ase – Yes-1 – that is novel in HSC or AML biology. Knockdown of Yes-1 forced exit from G0 in HSCs and knockout of Yes-1 in murine HSCs enforced the transition from LT- to ST-HSCs and a gradual loss of transplantability, while overexpression of Yes-1 in human HSCs caused increased transplantability and enhanced retention of HSCs in G0. Our results implicate a novel kinase-family gene as a crucial regulator of the transition between long-term and short-term HSCs and provide a target for therapeutically manipulating this transition for transplantation.
6
Bone Scaffold Architecture Affects Development of
Bone Grafts from Human Embryonic Stem Cells
1Iván Marcos Campos, 1,2Darja Marolt, 1Petros Petridis,
1Sarindr Bhumiratana, 3Daniel Schmidt, 1Gordana
Vunjak-Novakovic.
1Department of Biomedical Engineering, Columbia University, New
York, USA. 2NYSCF, New York, NY, 3University of Massachusetts at
Lowell, Boston, MA
Bone grafts are frequently needed to replace tissue extensively damaged by trauma or disease. Autologous grafts are limited and usually require painful surgical procedures causing donor site morbidity. As a promising alternative, tissue engineers strive to generate viable bone-like tissue substitutes, with similar functional properties to native bone. Previous studies using decellularized bovine bone scaffolds confirmed the osteogenic properties of this material, and its compositional, structural and biomechanical similarity to native human bone matrix. However, differences in scaffold architecture exist even among identical harvesting sites. In this work we study the influence of the scaffold architecture (density, pore size, porosity, surface area) and demineralization on osteogenic differentiation of human embryonic stem cells (hESC) and the deposition of mineralized bone matrix.
7
Conclusion: Our study reveals for the first time that internal architecture of decellularized bone scaffolds affects proliferation and osteogenesis of hESC-derived mesodermal progenitors. Different patterns of bone-like tissue deposition in scaffold density groups, and in particular reduced tissue formation in high density scaffolds, suggests the need for scaffold optimization to achieve homologous graft development in vitro.
7
Mir-17~92
cluster:
a
double-edged
sword
controlling spinal motor neuron generation and
degeneration
Jun-An Chen
1and Hynek Wichterle
1,*Departments of Pathology and Cell Biology, Neurology, and
Neuroscience, Center for Motor Neuron Biology and Disease,
Columbia Stem Cell Initiative, Columbia University Medical Center,
New York, NY, USA.
MicroRNAs (miRNAs) are small non-coding RNAs that function during embryonic development and in pathological process such as tumor formation. A representative example is the polycistronic mir-17~92 cluster known to have dual roles in normal B-cell development and cancerous B-cell lymphomas. Recently we have demonstrated that mir-17~92 cluster refines ventral spinal cord patterning during neural development, and its loss results in an increase in the number of newborn motor neurons. Here we report that the mir-17~92 cluster is also strongly expressed by postmitotic motor neurons. Surprisingly, analysis of mir-17~92 null motor neurons in vitro and in vivo revealed that its expression is critical for motor neuron survival. Null spinal cord exhibits an overall decrease in the number of motor neurons despite their initial overproduction. Moreover, overexpression of mir-17~92 in the spinal cord can prevent naturally occurring cell death. Currently we are investigating whether deregulation of mir-17~92 cluster might contribute to motor neuron loss in degenerative diseases such as ALS or SMA.
8
Prospective identification and purification of
quiescent and activated adult neural stem cells
from their in vivo niche
Paolo Codega, Violeta Silva-Vargas, Erika Pastrana, Angel R
Maldonado-Soto and Fiona Doetsch
Quiescent neural stem cells are thought to support adult neurogenesis throughout life, however their in vivo identity has been elusive. Here, using a combination of GFAP, CD133 and EGFR, we identify quiescent and activated stem cell astrocytes in the adult subventricular zone (SVZ) and define a strategy that allows their simultaneous prospective purification. Activated stem cell astrocytes are highly proliferative in vivo and constitute the major neurosphere-forming population in vitro. In contrast, quiescent stem cell astrocytes, contained in two populations, are largely dormant in vivo and only rarely form neurospheres, even during regeneration. However, once activated, they are highly proliferative, self-renewing and multipotent. Conversely, activated populations can revert to a more quiescent state in vitro. These findings yield insight into the heterogeneity of astrocytes within the SVZ stem cell niche, provide a powerful approach for defining their functional properties in vivo and in vitro and will allow elucidation of their intrinsic and extrinsic regulation.
9
Extracellular
Matrix
Derived
from
Kidney
Regulates the Growth and Metabolism of Kidney
Stem Cells with Regional Specificity
J. O'Neill, A. Anandappa, D. O. Freytes, J. A. Oliver, and G.
Vunjak-Novakovic
Introduction: Extracellular matrix (ECM) provides cells an ideal scaffold with tissue-specific cues (molecular, structural, biomechanical) that mediate cell
function. Stem cells are known to reside in specialized ECM niches where they remain quiescent until needed, such as the stem cells in the papilla region of the kidney. Currently, it is not possible to re-create the complex extracellular microenvironment of the kidney using synthetic materials. Therefore, our objective was to derive decellularized ECM sheets from the three regions of the kidney – cortex, medulla, and papilla – to investigate any region-specific effects on the growth and metabolism of kidney stem cells (KSCs) as well as non-native mesenchymal stem cells (MSCs).
Methods: Mouse MSCs were purchased, and KSCs were isolated from the renal papilla of mouse kidneys. Porcine kidneys were sectioned to separate cortex, medulla, and papilla regions. Each region was decellularized by serial washes in 0.02% trypsin, 3% Tween, 4% deoxycholic acid, and 0.1% peracetic acid solutions. ECM sheets were obtained using a 7mm biopsy punch. KSCs and MSCs were cultured on kidney region ECM sheets. Metabolic activity, DNA quantification, and confocal imaging were performed.
Results/Discussion: KSCs cultured on decellularized papilla ECM showed higher metabolic activity and lower DNA content when compared to KSCs on cortex and medulla ECM (left), an effect not observed with MSCs (right). Differences in KSC morphology, alignment, and structure formation were also observed via confocal imaging between all three regions (not observed with MSCs). These results suggest that decellularized kidney region ECM sheets provide region-specific molecular and topographical cues for KSCs that are not recognized by MSCs.
Conclusions: Decellularized kidney ECM affects the growth and metabolism of KSCs with regional specificity. Region-specific ECM may thus provide an optimal substrate for the cultivation and delivery of stem cells and their derivatives.
10
Expanding the Synthetic Capabilities of the Cell
Virginia Cornish Group
Synthetic Biology aims to engineer artificial pathways in cells both to test fundamental notions of how complex biological networks function and to develop new technologies for the treatment of disease. Moving beyond advances in the last century for the synthetic manipulation of biomolecules in vitro, our laboratory is creating conceptually new approaches for the modification of biomolecules in a living cell. As opposed to designing molecules that can be added to the cell with minimal perturbation of its natural functions, our approach is to design molecules capable of co--‐opti g the ell s o highl
evolved and integrated synthetic machinery (evolution and translation) for production of the modified biomolecules in vivo. Finally, we are using these new synthetic methods to develop tools for live cell imaging.
11
Deciphering the contribution of endothelial cells
and pericytes in the SVZ neural stem cell niche
Elizabeth E Crouch, Chang Liu, Fiona Doetsch
8
brain pericytes exhibit properties of mesenchymal stem cells (MSC), a cell type not known to exist in the adult mammalian brain. MSCs derived from SVZ pericytes are more osteogenic and chrondrogenic in vitro than either cortically-derived MSCs or bone-marrow cortically-derived MSC, but less adipogenic in vitro than bone-marrow derived MSCs. These studies illustrate how purifying primary perivascular cells from a stem cell niche can uncover novel players and roles in adult neural stem cell biology.
In stratified epithelial tissues, homeostasis relies on the self- renewing capacity of stem cells located within the innermost basal layer. However, signaling events that orchestrate the exit of basal progenitor cells from their niche, and epithelial stratification, remain to be identified. Here we report that phosphoinositide- dependent kinase 1 (PDK1) deficient skin lacks both epithelial differentiation and stratification. PDK1KO keratinocytes do not undergo calcium-induced phosphorylation of AKT, GSK3beta and atypical PKC, and fail to differentiate. There were also very few asymmetric divisions of basal cells in PDK1KO skin. PDK1KO epidermis shows decreased Notch expression and restoration of Notch3 rescued this differentiation defect. These results suggest that PDK1 is a key molecule that governs epithelial cell differentiation and stratification through its role in asymmetric cell division.
13
Engineering bone tissue from human embryonic
stem cells
Darja Marolt
1,2, Ivan Marcos Campos
1, Sarindr Bhumiratana
1,
Ana Koren
1,3, Petros Petridis
1, Geping Zhang
4, Patrice F.
Spitalnik
4, Warren L. Grayson
5and Gordana Vunjak Novakovic
11
Department of Biomedical Engineering, Columbia University;
2The
New York Stem Cell Foundation;
3Blood Transfusion Centre of
Slovenia;
4Department of Pathology and Cell Biology, College of
Physicians and Surgeons, Columbia University;
5Department of
Biomedical Engineering, School of Medicine, Johns Hopkins University.
Bone tissue engineering could provide an unlimited supply of functional bone grafts for surgical treatment of large bone defects. Pluripotent stem cells represent a promising cell source for this goal, as they can grow indefinitely, providing unlimited numbers of tissue repair cells, and give rise to any cell type in the body. In prior studies, limited formation of bone tissue was observed from human embryonic stem cells (hESC), accompanied by the development of teratomas. The goal of our study was to establish feasibility of engineering fully viable, ~0.5 cm large compact bone constructs, and to evaluate their phenotypic stability and safety in vivo.We developed a stepwise engineering protocol, where hESC were first induced into progenitors expressing mesenchymal surface markers and in vitro differentiation potential into osteogenic, chondrogenic and adipogenic lineages. Cultivation of hESC-mesenchymal progenitors in osteoconductive scaffolds in perfusion bioreactors for 5 weeks resulted in compact tissue constructs with significantly higher cell numbers, alkaline phosphatase activity, osteopontin release and dense bone matrix compared to static cultures.
Engineered bone tissue matured during the 8 weeks of subcutaneous implantation, resulting in denser bone matrix compared to scaffolds seeded with hESC-mesenchymal progenitors prior to implantation. Further maturation of the mineralized matrix was detected by micro-CT imaging. There was no evidence of teratoma formation. In addition, engineered bone constructs contained microvasculature spanning the interior regions of the scaffolds, and osteoclasts in the outer regions, suggesting initiation of tissue remodeling. Our results demonstrate hESC-mesenchymal progenitors can be induced to form compact, homogenous and phenotypically stable bone-like tissue in controllable fashion, by cultivation on three-dimensional osteoconductive scaffolds in bioreactors with interstitial flow of culture medium. We propose
that engineering bone-like tissue from human pluripotent cells can help advance fundamental study of osteogenesis, as well as translation into regenerative medicine applications.
14
Statins Promote Axonal Regeneration in the
Presence of Myelin
Hai Li
1, Elena Nikulina
3, Jianwei Hou
3, Wan Seok Yang
1,2,
Timothy Spencer
1, Gist Croft
1, Hynek Wichterle
1, Marie T. Filbin
3,
Brent Stockwell
1,2, Christopher E. Henderson
11
Columbia University, Center for Motor Neuron Biology and Disease,
New York, New York
2
Howard Hughes Medical Institute, Department of Biological Sciences
and Department of Chemistry, Columbia University, New York, NY
3
Biology Department, Hunter College, City University of New York,
New York, New York
Many factors contribute to the poor regeneration of adult mammalian CNS axons: lack of trophic support, inhibitory molecules associated with myelin and formation of glial scars. Some small molecules, such as cAMP or ROCK inhibitors, have been reported to stimulate neuronal growth on myelin. Although these compounds provide important insights into mechanisms of regeneration, there have been few direct attempts to identify more drug-like compounds with axon growth-promoting activity. We developed a high-throughput neuron-based assay to screen for compounds that enhance axonal regeneration on inhibitory substrata. Embryonic stem cell-derived motor neurons (ES-MNs) were cultured on monolayers of myelin-associated-glycoprotein-(MAG)-expressing CHO cells, and 50,000 compounds were tested for their ability to overcome MAG inhibition of neurite outgrowth. Unexpectedly, among the most effective compounds were statins. Cerivastatin, the most potent, induced a >3-fold increase in neurite growth with an EC50 of 35
nM. Statins inhibit HMG-CoA reductase (HMGCR), which converts 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) to mevalonate, an early rate-limiting step in cholesterol biosynthesis. HMGCR is highly expressed within the CNS. Two arguments suggest that HMGCR is the relevant target for the effects we observe. First, the rank order of potency of statins in our assay mirrors that for HMGCR inhibition in vitro. Second, the statin-induced increase in axonal regeneration can be reversed by addition of mevalonate. Thus, inhibition of HMG-CoA reductase and downstream pathways might constitute a therapeutic target in conditions such as spinal cord injury and ALS in which axonal regrowth needs to be stimulated.
15
Columbia
genome
center
high
throughput
screening and chemistry shared core facility for
NYSTEM users
Andrea Califano
1, Christopher E. Henderson
2, Charles Karan
3,
Hai Li
3, Sergey Pampou
3,
1
Department of Biomedical Informatics;
2Departments of Pathology,
Neurology and Neuroscience;
3Columbia Genome Center; Columbia
University Medical Center
The Columbia University Genome Center High Throughput Screening Center was established to allow Columbia University and NYSTEM researchers access to state of the art laboratory automation and detection technologies. This
auto atio , o i ed ith the e pertise of the e ter s staff, is desig ed to
9
and a GE IN Cell Analyzer 2000 for automated microscopy. In accordance with its mission, the center has identified key personnel and identified equipment essential to the fulfillment of its mission. The establishment of a screening center which can provide access to both small molecules and biological reagents will aid in the prosecution of new targets and provide new insights in the treatment of disease.
For more information on services available from the screening center contact
screening@columbia.edu.
16
Identification of Master Regulators of Pluripotency
and Lineage-Specific Differentiation for Mouse
Epiblast Stem Cells
Hui Zhao
1,2,3, Mariano Alvarez
1,4,5, Celine Lefebvre
1,4,5, Andrea
Califano
1,4,5and Michael M. Shen
1,2,3.
1
Herbert Irving Comprehensive Cancer Center,
2Department of
Medicine,
3Department of Genetics and Development,
4Joint Centers
for Systems Biology,
5Department of Biomedical Informatics,
Columbia University Medical Center, New York, NY 10032.
Mouse Epiblast Stem Cells (EpiSCs) are pluripotent embryonic stem cells that behave more like Human Embryonic Stem Cells rather than mouse Embryonic Stem cells (mESCs). Deriving from early post-implantation embryos, EpiSCs represent a pivotal stage between naïve mESCs and more differentiated somatic cell types. However, the full spectrum of the genetic regulatory network of EpiSCs, containing master regulators of self-renewal, pluripotency, and lineage-specific differentiation, has not been completely identified. In this study, we are seeking to systematically analyze the regulatory network of EpiSCs and to identify novel master regulators. Our study will not only provide new understanding of the fundamental regulatory network of stem cells, but also provide novel insights into new methods of reprogramming as well as transdifferentiation.
We have employed an unbiased system biology approach to construct a draft EpiSC Interactome. We have also performed seven time-course experiments of EpiSCs which were treated with control medium, BMP4, retinoic acid, or SB431542, induced to form embryoid bodies, or differentiated into mesoderm or definitive endoderm. Samples were collected at a range of time points, and then gene expression signatures were produced from 216 samples by RNA-seq.
Usi g the MA‘INA algorith as ell as irtual proteo i s, e ha e a al zed
1,393 transcriptional factors represented in the draft interactome to identify novel master regulators. We have identified new candidate lineage-specific master regulators as well as 124 master regulators (FDR<0.01, including Nanog and Oct4) that exhibit similar pattern among all treatments. In our current efforts, we are pursuing the functional validation of these master regulators using lentiviral gain- and loss-of-function approaches.
17
Spatial enhancement of ATP fluorescence In
pulmonary epithelium by stem cell-donated
mitochondria
Mohammad N. Islam, Li Sun, Shonit R. Das and Jahar
Bhattacharya,
Lung Biology Laboratory, Pulmonary, Allergy & Critical Care,
Department of Medicine, Columbia University Medical Center, New
York, NY 10032
RATIONALE. Exogenous administration of bone marrow-derived mesenchymal stem cells (MSCs) is protective in sepsis-induced acute lung injury (ALI). However, the protective mechanisms are not entirely clear and are of considerable interest. In septic lungs, loss of mitochondrial function, hence cellular ATP leads to loss of alveolar function. We considered that MSCs might protect by transferring mitochondria to alveolar epithelium, thereby rescuing alveolar bioenergetics and restoring critical ATP-dependent lung function such as surfactant secretion.
METHODS. In anesthetized mice, we airway instilled lipopolysaccharide (LPS, 1
mg/kg), or buffer (control), followed 4 hours later by instillation of MSCs (2x105
cells). The MSCs expressed mitochondria-targeted fluorescent protein, DsRed. After 24 hours, we viewed alveoli by confocal microscopy of isolated, blood-perfused mouse lungs held at constant pulmonary artery, left atrial and alveolar pressures of 10, 3 and 5 cmH2O, respectively. To determine ATP content at the cellular level, we transfected alveoli with a GFP-expressing, ATP probe (Perceval). To quantify inflation-induced surfactant secretion, we imaged loss of lamellar body fluorescence in type 2 epithelial cells of intact alveoli (Ashino, 2000).
RESULTS. In alveoli of LPS-treated lungs translocation of MSC-derived mitochondria was evident by the localization of DsRed fluorescence in alveolar epithelium. No mitochondrial transfer occurred in control lungs. We confirmed transfection of the ATP probe by in situ immunofluorescence of GFP as well as through immunoblots for GFP in the whole lung homogenate. LPS decreased ATP fluorescence in the alveolar epithelium by 28±12% of control. This effect was completely abrogated following MSC instillation (p<0.05). Within 4 hours of MSC instillation, AE GFP fluorescence progressively increased. Mitochondrial transfer in just one segment of the alveolar wall was sufficient to enhance ATP fluorescence throughout the same alveolus as well as in adjacent alveoli. Hyperinflation-induced surfactant secretion, an ATP-dependent response, was blocked in LPS-treated lungs. By contrast, after MSC instillation, surfactant secretion was rescued in MSC-containing alveoli as well as in their neighboring alveoli (p<0.05).
CONCLUSIONS. We conclude from these findings (1) that MSCs transferred viable mitochondria capable of generating ATP to alveolar epithelium of endotoxin-treated lungs, but not of control lungs; (2) that the mitochondrial transfer rapidly increased alveolar ATP; (3) that these effects rescued surfactant secretion. We propose that mitochondrial transfer from MSCs to the alveolar epithelium of septic lungs might underlie the protective mechanism of MSCs in ALI (support HL 64896).
In recent years, generation of patient-specific induced pluripotent stem cells (PS-iPSCs) has been reported, which are useful tools to investigate unknown disease mechanisms and therapeutic testing. Moreover, autologous PS-iPSCs have tremendous potential to provide a source of cells for regenerative therapies for specific human diseases, because they have unlimited proliferative capacity and extensive differentiation capability into required cell types. We are focused on developing iPSC-based cell therapies for recessive dystrophic epidermolysis bullosa (RDEB) by directing their differentiation into skin keratinocytes and fibroblasts, which both produce type VII collagen. We previously demonstrated that normal iPSCs and RDEB PS-iPSCs can be differentiated into functional keratinocytes. However, the methodology for generating skin FBs from iPSCs has not been defined. Here, we aimed to develop a protocol for differentiation of skin FBs from iPSCs by first generating spindle-shape cells via embryoid body formation, and then using ascorbic acid and tra sfor i g gro th fa tor β TGFβ , hi h a e ha e esoder
10
19
A comprehensive on-line distance-learning course
centered on the many aspects of stem cell research
Submitted: April 6, 2011
Ruth L. Fischbach, PhD, MPE, Professor of Bioethics and Director,
Center for Bioethics, Columbia University and Mailman School of
Public Health
John D. Loike, PhD, Research Scientist, Physiology & Cellular
Biophysics, and Director of Special Projects, Center for Bioethics,
Columbia University
Janet Mindes, PhD, Consultant, Center for Bioethics, Columbia
University
Under the auspices of the Center for Bioethics and the Department of Biological Sciences, Columbia University, and funded by a grant from the New York State Stem Cell Initiative (NYStem), the authors (Fischbach, Loike, and Mindes) developed a comprehensive on-line distance-learning course centered on the many aspects of stem cell research, the first at Columbia. The on-line course is entitled Stem Cells: Biology, Bioethics, and Applications.
Our online course was developed to accompany a new undergraduate level biology course that features a comprehensive overview of stem cell research. Both on-line and undergraduate courses were developed and implemented with funding from the New York State Stem Cell Initiative (NYStem), under the
‘FA, De elop e t a d I ple e tatio of College a d U i ersit Curri ula
Concerning Stem Cell Science and Related Ethical, Legal, and Societal
I pli atio s.
The on-line course was created by Drs. Ruth L. Fischbach, John D. Loike and Janet Mindes. The Faculty for the undergraduate course includes Professors Daniel Kalderon (PI), Carol Lin, Ruth Fischbach, and John Loike.
The course consists of 8 Modules on the major topics of stem cell science (see below). These are accompanied by 6 Supplements that enrich the science with innovative interdisciplinary discussion of topics such as the legal and political history of stem cell science, ethical considerations of egg donation and of therapeutic and reproductive cloning, animal welfare in the context of stem cell research, stem cell tourism, and the ethical conduct of clinical research. The on-line distance-learning course will be available as of May 7, 2012. We hope the course will become a resource for the Columbia University stem cell research community.
The course authors welcome feedback from members of the Columbia Stem Cell Initiative (CSCI) and are eager to collaborate with the CSCI, particularly concerning bioethical issues raised by stem cell research.
The course can be accessed at:
http://stemcellbioethics.wikischolars.columbia.edu/
The Center for Bioethics has created other notable on-line bioethics courses including Conflicts of Interest in Research, Privacy and Confidentiality, and Neuroethics: Implications of Advances in Neuroscience (http://www.cumc.columbia.edu/dept/bec/education/distance_learning.html).
20
The Msx1 Homeoprotein Differentially Recruits
Histone Methyltransferases to Repressed Target
Genes in Myogenic Progenitor Cells
Jingqiang Wang and Cory Abate-Shen
Departments of Urology and Pathology & Cell Biology, Herbert Irving
Comprehensive Cancer Center, Columbia University, College of
Physicians and Surgeons, New York, NY 10032.
Although the significance of lysine modifications of core histones for regulating gene expression is widely appreciated, the mechanisms by which these modifications are incorporated at specific regulatory elements during cellular differentiation remains largely unknown. We now show that genomic binding by Msx1 promotes enrichment of the H3K9me2 and H3K27me3 marks on repressed target genes via recruitment of G9a and Ezh2 histone methyltransferases, the enzymes responsible for catalyzing these histone marks
respectively. Interaction of Msx1 with G9a and Ezh2 is mediated via the homeodomain and is required for transcriptional repression and regulation of cellular differentiation, as well as enrichment of the H3K9me2 and H3K27me3 marks in proximity to Msx1 binding sites on repressed target genes in myoblast cells as well as the developing limb.Based on our findings on the recruitment of the H3K9me2 and H3K27me3 marks to target genes, we describe four distinct categories of Msx1 target genes that are distinguished by differential recruitment of the relevant histone methyltransferases. Our findings suggest that an important means of regulating gene expression during development involves the differential recruitment of histone modifying enzymes to the regulatory regions of target genes to influence chromatin status.
21
Flt3 is required for MLL-ENL-induced
leukemo-genesis in vivo, but not for LSC function in vitro
Kenjiro Kamezaki, Hans-Willem Snoeck
Columbia Center for Translational Immunology, Columbia University
Medical Center, New York, NY
11
22
Distinct mechanisms trigger maturation and
reactivity of spinal cord astrocytes derived from
human pluripotent stem cells
Laurent Roybon
1,2, Nuno J. Lamas
1,2,*, Alejandro D. Garcia
1,*, Eun
Ju Yang
3, Rita Sattler
3, Vernice J. Lewis
2, Yoon A. Kim
1, C. Alan
Kachel
1, Jeffrey D. Rothstein
3, Serge Przedborski
2, Hynek
Wichterle
1,2and Christopher E. Henderson
1,2.
1. Project A.L.S./Jenifer Estess Laboratory for Stem Cell Research, New
York, NY 10032, USA.
2. Departments of Pathology, Neurology and Neuroscience, Center for
Motor Neuron Biology and Disease (MNC), and Columbia Stem Cell
Initiative (CSCI), Columbia University Medical Center, New York, NY
10032, USA.
3. Department of Neurology, Institute for Basic Biomedical Sciences,
Johns Hopkins University, Baltimore, Maryland, USA.
* These authors contributed equally.
Astrocytes show a high degree of molecular and functional heterogeneity throughout development and in the healthy and diseased adult central nervous system but this is only partly modeled by current in vitro systems. We sought to define the cellular mechanisms underlying maturation and reactivity in mouse and human pluripotent stem cell-derived astrocytes. Using new accelerated protocols for differentiation of ES and iPS cells we showed that the resulting astrocytes were immature by both biochemical and functional criteria, even when grafted in mouse brain. Remarkably, short treatments with single factors were able to selectively direct these spinal cord astrocytes toward mature or
rea ti e phe ot pes, respe ti el . FGF , ut ot TNFα, as suffi ie t to i du e arkers of ature astro tes a d full fu tio al a ti it . Co ersel TNFα, ut
not FGF1, induced multiple elements of a reactive phenotype but did not affect maturation. These phenotypically defined, scalable populations of mouse and human astrocytes will be important both for analysis of normal astrocyte function and for modeling human pathological processes in vitro.
23
Stem cell-based approaches to the neurobiology of
obesity in Bardet-Biedl Syndrome
Liheng Wang
1,2,3, Dieter Egli
2, Damian Williams
4, Stephen H.
Tsang
5, Robin S. Goland
1, Amy B. MacDermott
4, Rudolph L.
Leibel
1,31
Division of Molecular Genetics and Naomi Berrie Diabetes Center,
Columbia University College of Physicians and Surgeons, New York,
NY 10032.
2
New York Stem Cell Foundation, 3960 Broadway, New York, NY
10032.
3
Institute of Human Nutrition, Columbia University, New York, NY
10032.
4
Department of Physiology & Cellular Biophysics, Columbia
University, New York, NY 10032.
5
Department of Ophthalmology, Columbia University, New York,
NY10032.
Bardet-Biedl Syndrome (BBS) is an autosomal recessive genetic disease, which is characterized by polydactyly, obesity, retinal degeneration, renal cysts and mental retardation. BBS is caused by mutations in a group of proteins that are components of the basal body of the primary cilium. Bbs null mice display impaired leptin receptor signaling and reduced number of hypothalamic POMC neurons. Leptin and insulin signaling are two key hypothalamic pathways involved in regulating energy homeostasis, in part through effects on the activity of POMC neurons in the hypothalamus and brainstem. To investigate the neurobiology of obesity in BBS, we reprogrammed skin fibroblasts from two BBS patients carried the BBS1M390R/M390R and BBS10 C91fsX95/ C91fsX95 mutation respectively, into induced pluripotent stem cells (iPSCs) and further
differentiated them into insulin or leptin sensing neurons. iPS cells derived from unaffected healthy subjects are used as control in this study. We first demonstrated that the ciliogenesis was not affected in BBS1 mutant iPSC-derived neurons while BBS10 mutant neurons displayed longer cilia. Furthermore, the insulin-induced AKT phosphorylation at both T308 and S473 sites was greatly reduced in both BBS1 and BBS10 mutant neurons compared to control neurons. Leptin signaling was investigated in BBS mutant fibroblasts expressing exogenous LEPR, and we found that both BBS mutations impaired leptin mediated STAT3 activation. These data suggest that mutation of BBS proteins can affect both leptin and insulin signaling, which may contribute to obesity in BBS patients.
(Supported by NYSCF; NYSTEM Contract#026184; Rudin Foundation and Russell Berrie Foundation.)
24
PDGFRβ in the adult SVZ stem cell lineage
Angel R. Maldonado-Soto, Paolo Codega, Alex Paul, Annina M.
DeLeo and Fiona Doetsch
Abstract: Specific regions within the adult mammalian brain maintain the ability to generate neurons. The largest of these, the subventricular zone (SVZ), comprises the entire lateral walls of the lateral ventricles. Here, a subset of glial fibrillary acid protein (GFAP)-positive astrocytes (B cells) gives rise to neurons and oligodendrocytes throughout life. This process of neurogenesis involves quiescent B cells becoming proliferative (epidermal growth factor receptor (EGFR)-positive) and giving rise to neuroblasts via transit amplifying precursors. Studying the more quiescent cells in this niche has been hindered by the lack of molecular markers to selectively target them. Using microarray and qPCR analysis of putative quiescent neural stem cells (qNSCs) we determined that they were enriched for the PDGF‘β mRNA. We used immunostaining to determine the in vivo identity of PDGF‘β-positive cells, and discovered that only GFAP+ cells within the SVZ stem cell lineage express PDGF‘β. ThesePDGF‘β+ B ells o ta t the e tri le at the e ter of
ependymal pinwheel structures. Moreover, the vast majority of the PDGF‘β+ B
cells were EGFR- and slowly dividing in vivo. Detailed morphological analyses of these cells revealed primary cilia at their apical process in contact with the ventricle and long radial processes contacting blood vessels deep within the SVZ, both of which are characteristics of adult neural stem cells. Finally, we developed a strategy using fluorescence-activated cell sorting (FACS) to purify PDGF‘β+ astro tes a d sho that i itro they are capable of self-renewal and are multipotent. Together these findings indicate PDGF‘β+ B ells
are quiescent stem cells. Ongoing studies are aimed at performing lineage tracing of these cells to determine their in vivo progeny, as well as further dissecting the role of the PDGF‘β in the SVZ stem cell niche.
25
Retinoids are required in a novel progenitor
population for formation and regeneration of the
urothelium.
Andrei Molotkov, Devangini Gandhi, Maia Reiley, Kerry
Schneider and Cathy Mendelsohn
Our studies are focused on identifying the progenitor cells that control differentiation and regeneration of the urothelium, a stratified epithelium that lines the urinary outflow tract, extending from the renal pelvis to the urethra. This specialized epithelium is water-resistant and is crucial for preventing exchange of toxic substances between the blood and urine. It is lined by a layer of large, multinucleated umbrella cells that synthesize and secrete uroplakins, a family of proteins that assemble into crystalline plaque that generates the urothelial barrier.
