Fischbach RL Loike JD Mindes J 2011 . Ab

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

4:00 PM

Townhall meeting - CSCI strategy, Recruitment, Facilities, Training program

Moderated by Chris Henderson

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

Despite recent advances in basic and translational research, prostate cancer remains the second leading cause of cancer in men and a complete cure remains elusive. Complications in the research field arise from a lack of research tools designed to investigate the early events of human prostate tumorigenesis. To address this, we focused our study on reprogramming and lineage conversion of readily-accessible fibroblasts into prostate epithelial cells with the purpose of creating a new methodology for human neoplastic prostatic tissue modeling. We show here that mouse embryonic fibroblasts can be directly converted into epithelial cells in culture following expression of reprogramming factors, in the absence of an intermediate pluripotent stage. Furthermore, expression of known master regulators of prostate development is able to determine complete differentiation of the induced epithelial cells into prostatic tissue in vivo in tissue recombination assays. These studies represent the initial steps of an ambitious novel methodology for the generation and functional analysis of patient-specific normal and transformed prostate tissue using in vivo methods, which would have considerable clinical relevance.

Posters

Heart Center Riverview Terrace

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.

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 1,2

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, 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., P 2 ALS, NYSTEM contract #C024415 , NIH GO grant 5

RC2 NS069395-02

Defining the fate of ghrelin-expressing cells in pancreas

Luis Arnes 1 , Jonathon Hill 1 , Mark Magnuson 2 and Lori Sussel 1

1 Dept. of Genetics and Development, Columbia University, New York, NY 10032; 2 Department 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 ell type 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. Our analysis shows that a large number of the PP cells in the adult islet are descendants of the embryonic ghrelin cells, in addition to a subpopulation of the glucagon-producing cells. Notably, we also observed ghrelin descendants co-staining with exocrine and ductal markers, suggesting that there is a population of ghrelin-expressing cells that is not uniquely committed to the endocrine lineage. Supporting this hypothesis ghrelin-expressing cells are present in the absence of Ngn3, the endocrine progenitor population marker. Although the number of PP cells is decreased in Nkx2.2 null pancreas, a subpopulation of remaining PP cells are also marked with the ghrelin lineage- label, suggesting that Nkx.2.2 is not essential for the transition of ghrelin- expressing cells to PP-expressing cells. Moreover, although ghrelin cells are upregulated in Nkx2.2 null, we did not see an increase in the number of acinar or duct cells labeled with the tracer, suggesting that this population of ghrelin cells is also independent of Nkx2.2.

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

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.

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.

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. Material and Methods: Bovine bone scaffolds (4 mm in diameter x 2 mm in thickness) were sorted into three experimental groups according to density: low den¬sity (0.25-0.30 mg/mm3), medium density (0.425-0.475 mg/mm3) and high density (0.60-0.65 mg/mm3). For the assessment of the influence of native bone matrix composition on osteogenic differentiation, medium density scaffolds were fully demineralized. Bone internal architecture was determined for each group by measuring pore size, porosity, surface area and elastic compressive modulus. Mesodermal progenitors derived from hESCs were seeded on the scaffolds (6 x 105 cells/scaffold) and cultured for 5 weeks. DNA content, cell viability, bone deposition and osteogenic gene expression were quantified in engineered constructs at day 1, day 3, week 1, week 3 and week 5. Results and discussion: Analysis of the internal architecture of decellularized bone showed decreasing pore size, porosity and increasing surface area and mechanical properties with increasing scaffold densities. Remarkable differences in pore size, porosity, surface area and mechanical properties were found among the groups. A live/dead assay revealed that cell viability was not significantly affected by scaffold architecture after 5 weeks. HE stainings demonstrated uniform cell distribution with thin cell layers covering all available surface areas 24h after seeding. By week 3, the low and medium density scaffolds exhibited a higher cell number and denser tissue growth in the internal void volume of the scaffold. In contrast, cell content of high-density scaffolds was considerably lower, with void areas in the scaffold interior. Moreover, cell proliferation in high-density scaffolds was found to be much slower compared to low density groups. Interestingly in all groups, the final cell density (as determined at week 5) was reached by 3 weeks of culture, suggesting that a saturation level had been achieved. Osteogenesis was demonstrated in all groups by the presence of osteopontin and bone sialoprotein, with most of the bone matrix formation observed in areas with high cell density, and with more abundant matrix formation in medium scaffold density group. We also found slight differences between demineralized and mineralized matrix scaffolds, suggesting that the extent of mineralization also plays a role in osteogenesis.

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.

Mir-17~92 cluster: a double-edged sword

controlling spinal motor neuron generation and degeneration

Jun-An Chen 1 and 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.

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.

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.

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.

Deciphering the contribution of endothelial cells and pericytes in the SVZ neural stem cell niche Elizabeth E Crouch, Chang Liu, Fiona Doetsch

Neural stem cells reside in specialized niches in the adult mammalian brain and continuously produce new neurons that integrate into neural circuits. The largest germinal region in the adult brain is the subventricular zone (SVZ), which lies adjacent to the lateral ventricles and generates olfactory bulb neurons. SVZ stem cells and their progeny reside in a specialized vascular niche. Using a novel fluorescence activated cell sorting (FACS) protocol, we are able to purify primary endothelial cells and pericytes directly from the SVZ and from two non- neurogenic regions, the cortex and striatum, in a fast and efficient manner. Analysis of these purified perivascular populations has revealed novel aspects about their in vitro behavior, transcriptional profiles, and effect on specific stages of the neural stem cell lineage. Endothelial cells isolated from the SVZ exhibit different growth properties in vitro than cortical endothelial cells. Additionally, endothelial cells from the SVZ, striatum and cortex have distinct molecular signatures as determined by microarray analysis. Despite these differences, endothelial cells from both SVZ and cortex promote dramatic proliferation of cells from activated stages of the neural stem cell lineage but do not activate quiescent neural stem cells in vitro. Very little is known about the heterogeneity of pericytes in the adult brain. Here, we identify previously undescribed subpopulations of pericytes based on cell surface antigens, size, morphology, and proliferation capacity in vitro. Additionally, subpopulations of Neural stem cells reside in specialized niches in the adult mammalian brain and continuously produce new neurons that integrate into neural circuits. The largest germinal region in the adult brain is the subventricular zone (SVZ), which lies adjacent to the lateral ventricles and generates olfactory bulb neurons. SVZ stem cells and their progeny reside in a specialized vascular niche. Using a novel fluorescence activated cell sorting (FACS) protocol, we are able to purify primary endothelial cells and pericytes directly from the SVZ and from two non- neurogenic regions, the cortex and striatum, in a fast and efficient manner. Analysis of these purified perivascular populations has revealed novel aspects about their in vitro behavior, transcriptional profiles, and effect on specific stages of the neural stem cell lineage. Endothelial cells isolated from the SVZ exhibit different growth properties in vitro than cortical endothelial cells. Additionally, endothelial cells from the SVZ, striatum and cortex have distinct molecular signatures as determined by microarray analysis. Despite these differences, endothelial cells from both SVZ and cortex promote dramatic proliferation of cells from activated stages of the neural stem cell lineage but do not activate quiescent neural stem cells in vitro. Very little is known about the heterogeneity of pericytes in the adult brain. Here, we identify previously undescribed subpopulations of pericytes based on cell surface antigens, size, morphology, and proliferation capacity in vitro. Additionally, subpopulations of

advance fundamental study of osteogenesis, as well as translation into pericytes are more osteogenic and chrondrogenic in vitro than either cortically-

regenerative medicine applications.

derived MSCs or bone-marrow 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.

Statins Promote Axonal Regeneration in the Presence of Myelin

12 Hai Li 1 , Elena Nikulina 3 , Jianwei Hou 3 , Wan Seok Yang 1,2 , PDK1 regulates keratinocyte differentiation

Timothy Spencer 1 , Gist Croft 1 , Hynek Wichterle 1 , Marie T. Filbin 3 , Teruki Dainichi 1 , Matthew S. Hayden

and Sankar Ghosh 1, 2 1 Brent Stockwell 1,2 , Christopher E. Henderson 1

1 Columbia University, Center for Motor Neuron Biology and Disease, Dermatology, Columbia University

1 Department of Microbiology & Immunology and 2 Dept. of

New York, New York

In stratified epithelial tissues, homeostasis relies on the self- renewing capacity

2 Howard Hughes Medical Institute, Department of Biological Sciences

of stem cells located within the innermost basal layer. However, signaling

and Department of Chemistry, Columbia University, New York, NY

events that orchestrate the exit of basal progenitor cells from their niche, and

3 Biology Department, Hunter College, City University of New York,

epithelial stratification, remain to be identified. Here we report that

New York, New York

phosphoinositide- dependent kinase 1 (PDK1) deficient skin lacks both epithelial differentiation and stratification. PDK1KO keratinocytes do not undergo

Many factors contribute to the poor regeneration of adult mammalian CNS calcium-induced phosphorylation of AKT, GSK3beta and atypical PKC, and fail to

axons: lack of trophic support, inhibitory molecules associated with myelin and differentiate. There were also very few asymmetric divisions of basal cells in

formation of glial scars. Some small molecules, such as cAMP or ROCK PDK1KO skin. PDK1KO epidermis shows decreased Notch expression and

inhibitors, have been reported to stimulate neuronal growth on myelin. restoration of Notch3 rescued this differentiation defect. These results suggest

Although these compounds provide important insights into mechanisms of that PDK1 is a key molecule that governs epithelial cell differentiation and

regeneration, there have been few direct attempts to identify more drug-like stratification through its role in asymmetric cell division.

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

13 neurons (ES-MNs) were cultured on monolayers of myelin-associated- glycoprotein-(MAG)-expressing CHO cells, and 50,000 compounds were tested

Engineering bone tissue from human embryonic

for their ability to overcome MAG inhibition of neurite outgrowth.

stem cells

Unexpectedly, among the most effective compounds were statins. Cerivastatin, the most potent, induced a >3-fold increase in neurite growth with an EC 50 of 35

Darja Marolt 1,2 , Ivan Marcos Campos 1 , Sarindr Bhumiratana 1 ,

nM. Statins inhibit HMG-CoA reductase (HMGCR), which converts 3-hydroxy-3-

Ana Koren 1,3 , Petros Petridis 1 , Geping Zhang 4 , Patrice F.

methylglutaryl coenzyme A (HMG-CoA) to mevalonate, an early rate-limiting Spitalnik 4 , Warren L. Grayson 5 and Gordana Vunjak Novakovic 1 step in cholesterol biosynthesis. HMGCR is highly expressed within the CNS.

1 Department of Biomedical Engineering, Columbia University; 2 The

Two arguments suggest that HMGCR is the relevant target for the effects we

3 New York Stem Cell Foundation; observe. First, the rank order of potency of statins in our assay mirrors that for Blood Transfusion Centre of HMGCR inhibition in vitro. Second, the statin-induced increase in axonal Slovenia; 4 Department of Pathology and Cell Biology, College of

regeneration can be reversed by addition of mevalonate. Thus, inhibition of

Physicians and Surgeons, Columbia University; 5 Department of

HMG-CoA reductase and downstream pathways might constitute a therapeutic

Biomedical Engineering, School of Medicine, Johns Hopkins University.

target in conditions such as spinal cord injury and ALS in which axonal regrowth Bone tissue engineering could provide an unlimited supply of functional bone

needs to be stimulated.

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

Columbia genome center high throughput

from human embryonic stem cells (hESC), accompanied by the development of teratomas. The goal of our study was to establish feasibility of engineering fully

screening and chemistry shared core facility for

viable, ~0.5 cm large compact bone constructs, and to evaluate their phenotypic

NYSTEM users

stability and safety in vivo.

We developed a stepwise engineering protocol, where hESC were first induced 1 Andrea Califano , Christopher E. Henderson 2 , Charles Karan 3 ,

into progenitors expressing mesenchymal surface markers and in vitro

Hai Li 3 , Sergey Pampou 3 ,

differentiation potential into osteogenic, chondrogenic and adipogenic lineages.

1 Department of Biomedical Informatics; 2 Departments of Pathology,

Cultivation of hESC-mesenchymal progenitors in osteoconductive scaffolds in

Neurology and Neuroscience; 3 Columbia Genome Center; Columbia

perfusion bioreactors for 5 weeks resulted in compact tissue constructs with

University Medical Center

significantly higher cell numbers, alkaline phosphatase activity, osteopontin The Columbia University Genome Center High Throughput Screening Center release and dense bone matrix compared to static cultures.

was established to allow Columbia University and NYSTEM researchers access Engineered bone tissue matured during the 8 weeks of subcutaneous

to state of the art laboratory automation and detection technologies. This implantation, resulting in denser bone matrix compared to scaffolds seeded

i ed ith the e pertise of the e ter s staff, is desig ed to with hESC-mesenchymal progenitors prior to implantation. Further maturation

auto atio , o

expedite new discoveries in stem cell science and provide a catalyst for new of the mineralized matrix was detected by micro-CT imaging. There was no

translational medicines. The center will allow users access to small molecules, evidence of teratoma formation. In addition, engineered bone constructs

which could provide new investigational leads for drug discovery, RNAi and contained microvasculature spanning the interior regions of the scaffolds, and

cDNA libraries, which can be used to better understand the underlying osteoclasts in the outer regions, suggesting initiation of tissue remodeling.

mechanisms at work in diseases through functional genomics assays. The Our results demonstrate hESC-mesenchymal progenitors can be induced to

center is comprised of multiple automated liquid handling systems, including a form compact, homogenous and phenotypically stable bone-like tissue in

Cell::Explorer automation system designed to handle large scale high controllable fashion, by cultivation on three-dimensional osteoconductive

throughput cellular assays. The center is also equipped with state of the art scaffolds in bioreactors with interstitial flow of culture medium. We propose

detection systems with a Perkin Elmer Envision for plate reader based assays detection systems with a Perkin Elmer Envision for plate reader based assays

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 1,4,5 and Michael M. Shen 1,2,3 .

1 Herbert Irving Comprehensive Cancer Center, 2 Department of Medicine, 3 Department of Genetics and Development, 4 Joint Centers

for Systems Biology, 5 Department 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.

Spatial enhancement of ATP fluorescence In pulmonary epithelium by stem cell-donated mitochondria