1. Generation of transgene-free iPSC lines from patients with Parkinson’s disease
Bi, K., Hermanson, S., Lebekken, C., Piekarczyk, M., Reichling, L., Barron, T., Vogel, K., Life Technologies, Madison, WI; Langston, J. W., Schuele, B., The Parkinson’s Institute and Clinical Center, Sunnyvale, CA.
Patient-derived induced pluripotent stem cells (iPSCs) offer exciting potential in both cell therapy and in vitro disease modeling. Efficient reprogramming of patient somatic cells to iPSCs plays a key role in realization of these potentials. Many reprogramming methods encounter technical challenges to convert adult/disease somatic cells to iPSCs consistently and with high efficiency. Methods that rely on integrating virus or plasmid to reprogram could potentially result in multiple insertions and risk of tumorigenicity. Reprogramming with episomal vectors, mRNAs and miRNAs has low reprogramming efficiency or requires multiple rounds of transfection and with specific types of cells. Sendai virus is a negative-strand RNA virus that replicates in the cytoplasm of infected cells and does not integrate into the host genome. Recent papers demonstrated that Sendai virus delivering the four Yamanaka factors is a highly efficient method to reprogram normal human foreskin fibroblasts, peripheral blood mononuclear cells and CD34+ cells to generate integration-free iPSCs. Here, fibroblasts from skin biopsies of four Parkinson’s disease (PD) patients and two age and gender-matched control individuals were efficiently reprogrammed to iPSCs using the Sendai reprogramming method. These iPSCs are transgene-free and karyotypically normal, express known pluripotency markers and are able to differentiate into embryoid bodies with three germ layers. Gene expression profiles clearly distinguished these iPSCs from their parental fibroblasts and clustered them with other iPSCs and H9 line. Given the efficiency, speed and ease with which we were able to reprogram adult disease fibroblasts, we anticipate Sendai reprogramming method being applied to large scale reprogramming of multiple disease lines potentially in an automated fashion.
2. IGERT Presentation: Creation of a human cell model of ataxia-telangiectasia for elucidation of epigenetic mechanisms underlying neurological disease symptoms
D’Ecclessis, M. (1), Gruenewald, A. (1), Toro-Ramos, A. (1), Swerdel, M.R. (1), Moore, J.C. (2,3), Hart, R.P. (1,3). 1 Department of Cell Biology & Neuroscience, 2 Department of Genetics, 3 Human Genetics Institute of NJ, RUCDR Infinite Biologics, Rutgers University, Piscataway, NJ
Ataxia-telangiectasia (A-T) is a rare, recessive genetic disorder caused by mutations in the ATM gene, which broadly impacts proper functioning of the nervous and immune systems. Mutations in the ATM gene have been shown to result in an Atm protein deficiency that reduces the ability to detect and repair DNA damage. Despite this knowledge, the cellular mechanism behind A-T and its neurological symptoms is less clear. It is hypothesized that epigenetic mechanisms are directly involved in the neurological symptoms of the disease, which include neurodegeneration and loss of neuronal function. We obtained blood samples from patients affected by A-T as well as from their parents who are carriers for the mutation. These blood samples were reprogrammed using Sendai viral vectors (Cytotune™) to create induced pluripotent stem cell (iPSC) lines that are heterozygous for ATM in the carrier line and compound heterozygous for ATM in the affected line since each inherited allele carries a different ATM mutation. By utilizing genome editing tools that include transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPRs), we are able to manipulate the ATM gene to produce additional human cell models of A-T. Furthermore, the resulting iPSCs can be differentiated into neurons in order to investigate the epigenetic mechanisms involved in the development of neurological A-T symptoms. We created a cell line that is heterozygous for a mutation in the ATM gene by replacing ATM exon 55 with a puromycin-resistance cassette. Exon 55 is significant because it encodes a portion of the functional kinase domain of Atm. The presence of this cassette and the proper location of its insertion have been experimentally verified by PCR. Together, these carrier, affected, and otherwise manipulated cell lines will serve as invaluable tools to better understand the cellular mechanisms behind the role of epigenetics in neurological symptoms of A-T.
Acknowledgements: Supported by the A-T Children’s Project. We thank Dr. Howard Lederman of the A-T Clinical Center at Johns Hopkins University for collecting specimens.
3. Development of Transgenic Zebrafish with Hematopoietic Stem Cell-specific Cre-mediated Recombination
Flaherty K.M., Davis S., and Sabaawy H.E.
Hematopoietic pathways are well conserved in zebrafish, making it an excellent model organism to study hematopoiesis and leukemia development. Hematopoietic stem and progenitor cells (HSPCs) self-renew and differentiate into multiple blood and immune cells throughout life. Primitive embryonic HSPCs exist briefly within the intermediate cell mass (ICM), while definitive HSPCs rise in the hemogenic endothelium of the dorsal aorta in all vertebrates including zebrafish. HSPCs are then released into the blood circulation to colonize the fetal liver in mammals, or the caudal hematopoietic tissue in zebrafish before reaching their final destination of the BM in mammals or kidney marrow in zebrafish. The ability to monitor the embryonic development of HSPCs, and perform lineage tracing of HSPCs and their progeny will be significantly enhanced by generating highly specific transgenic zebrafish reporters of HSPCs. Moreover, HSPCs may be the target cells associated with leukemia and lymphoma development. Therefore, identifying promoter elements that faithfully drive the expression of oncogenes in HSPCs, and can be utilized with the established Cre/Lox system for genetic recombination is essential for generating zebrafish leukemia and lymphoma models. We have used the mouse Runx1+24 kb intronic enhancer to generate GFP or DsRed reporter lines for HSPCs. Next, we utilized the same enhancer elements to generate a third inducible transgenic zebrafish line with the Runx1 enhancer driving Tamoxifen-inducible Cre (CreERt2) and a mCherry using Tol2 transgenesis. Zebrafish embryos with mCherry expression in the ICM at 2 days post-fertilization were raised to adulthood, and bred to wild-type fish to screen their F1 progeny for stable integration. Successful germ-line transmission was achieved establishing stable Runx1+24:mCherry-CreERt2 transgenic zebrafish line. When crossed to a complementary zebrafish that contain loxP sites and leukemia-specific oncogenes in the presence of Tamoxifen, CreERt2 expression targets recombination to HSPCs. This is the first transgenic zebrafish line with HSPC-specific CreERt2 expression. These zebrafish will allow for detailed HSPC lineage tracing studies, and a better understanding of the roles of oncogenes during leukemia development.
4. Dominant effect of muscular dystrophy embryonic stem cells in WT mice
JP Gonzalez1, J Schneider1, J Button1, C Chang1, S Kyrychenko2, V Kyrychenko2, J Doering3, M Bhaumik4, R Grange3, N Shirokova2 and D Fraidenraich1*. 1Departments of Cell Biology and Molecular Medicine and 2Pharmacology and Physiology, Rutgers-NJMS 3Department of Human Nutrition, Foods and Exercise, Virginia Tech 4Department of Pediatrics, Rutgers-CINJ * To whom correspondence should be addressed: firstname.lastname@example.org
We previously created chimeric mice by injecting WT mouse embryonic stem cells (ESCs) and induced pluripotent stem cells into mdx blastocysts, a model of Duchenne muscular dystrophy (DMD). Interestingly, a low percentage of pluripotent stem cells was sufficient to supply dystrophin to the heart and skeletal muscles and yield a significant amelioration of disease (Stillwell et al. and Beck et al., PLOS ONE 2009 and 2011). Recently, we generated mosaic mice by injecting mdx ESCs into WT blastocysts (termed ‘reverse’ chimeras). With low levels of ESC incorporation (10-30%), the mdx/WT chimeric heart acquired dystrophic features, in terms of intracellular calcium responses to mechanical stress, well before a typical mdx cardiac phenotype would appear. By separating and analyzing mdx ESC and WT blastocyst derived populations, we found that not only did ESC-derived mdx cardiac myocytes behave like typical pathological mdx cardiac myocytes, but blastocyst-derived WT cardiac myocytes also displayed typical mdx pathology. We also observed that at a higher degree of chimerism (30-50%), some skeletal muscles like the pectoralis and diaphragm, but not the quadriceps or soleus, showed histological features of muscular dystrophy. These affected muscles displayed non-uniform expression of dystrophin and compromised utrophin upregulation. Preliminary functional studies revealed that twitch, tetanus, and shortening velocities were diminished specifically in the EDL but not the soleus, again displaying a muscle-specific onset of pathology. In analyzing mature adipocytes, we observed an upregulation of skeletal and cardiac muscle markers as well as secreted hypertrophic factors such as Wnt5a and follistatin-like protein 1 (Fstl1), all normally found at higher levels in mdx mice compared to WT. Overall, these findings suggest that the ESC-derived mdx compartment defines the overall phenotype in the ‘reverse’ chimeric heart, some skeletal muscles, and adipose tissue. As mosaicism is a common feature in DMD symptomatic carriers and in Becker muscular dystrophy, this unsuspected dominant, muscle-specific function of the mdx ESC-derived cells merits further studies to potentially uncover therapeutic factors or pathways attributing to this phenomenon.
Acknowledgements: This work is supported by grants from the MDA and NIH. There are no conflicts of interest.
5. IGERT Presentation: NOS1AP stunts dendrite development and alters cytoskeleton dynamics in human neural cells
Kristina Hernandez 1, 2, Natasha R. Dudzinski 2, Vincent Luo 3, Jennifer C. Moore 4,5, Kenneth Paradiso 2, Michael Sheldon 4, 5, Jay A. Tischfield 4, 5, Ronald P. Hart 2, 4, Linda M. Brzustowicz 5 and Bonnie L. Firestein 2 1 Molecular Biosciences Graduate Program, 2 Dept. of Cell Biology and Neuroscience, 3 Dept. of Biomedical Engineering, 4 NIMH Stem Cell Resource, 5 Dept. of Genetics, Rutgers University, NJ
Proper excitatory neurotransmission is tied to dendritic arbor development as well as synapse formation and maturation. Many neurodevelopmental disorders show improper neuronal morphology, such as a reduction in dendrite number or branching. NOS1AP (nitric oxide synthase 1 [neuronal] adaptor protein), encoded by a schizophrenia susceptibility gene, is an intracellular factor that alters neuronal morphology and the composition of the postsynaptic density. Studies from the Firestein and Brzustowicz laboratories demonstrated that NOS1AP expression is upregulated in postmortem samples from the dorsolateral prefrontal cortex of patients with schizophrenia. To investigate the role that NOS1AP plays in human dendritic arbor development, we used human iPSC technology to generate human neural stem cells and neurons. We found that increased protein levels of NOS1AP decrease dendrite branching in human neurons at the developmental time point when both primary and secondary branching actively occurs. The initiation of a neurite requires the dynamic regulation of actin filaments and microtubules which results in the formation of an actin-rich protrusion followed by its invasion by bundled microtubules. To investigate the mechanism by which NOS1AP negatively alters dendrite branching we tested whether NOS1AP can alter actin polymerization, a process required for new branch points to occur. We show that increased levels of NOS1AP in COS-7 cells result in a decrease in polymerized actin, while total actin protein levels are unchanged. In addition, we find that NOS1AP can alter the organization of actin filaments in human neural stem cells. Together, these findings suggest that NOS1AP can regulate dendritogenesis in an activity independent manner by altering cytoskeleton dynamics.
Acknowledgements: This work was supported in part by NIH - NIMH, U24 MH068457. K. Hernandez was supported in part by NIH IMSD Grant No. 2R25 GM55145, "UMDNJ-Rutgers University Pipeline Program”; NIH Biotechnology Training Grant No. T32 GM008339-20; and NSF DGE 0801620, “IGERT: Integrated Science and Engineering of Stem Cells”.
6. Application of an intestinal organoid platform to identify colon cancer chemopreventive agents
Kuratnik, A., L. Cao, C. Nelson, C. Giardina, Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT Wright, D., Department of Pharmaceutical Sciences, University of Connecticut, Storrs, CT
Intestinal organoids are pseudo-organized multicellular structures that resemble intestinal epithelium and possess distinct zones of proliferation and differentiation. We have previously reported a two-step method that utilizes human and murine ESCs to derive intestinal organoids enriched for intestinal stem cells that resolve into distinct populations, as found in the colon and small intestine. We have also developed a high throughput approach for using intestinal organoids derived from cancer-prone ApcMin/+ murine cells as tissue surrogates for the study of the intestinal epithelium with a focus on identifying novel cancer-preventing compounds. An mRNA expression based screen of 90 epigenetically active compounds along with other smaller screens showed activation of cell differentiation pathways in a manner that resembled their functionality in vivo. We propose using this platform for the high throughput screening of novel compounds for potential cancer-preventive agents that regulate cell differentiation and proliferation pathways in the colon.
Acknowledgements: This work was supported by NCI R21 grant to CG.
7. Notch1 cis-element CR2 regulates gene expression in neural progenitors during spinal cord development
Ying Li, Evangeline Tzatzalos, Sung Tae Doh, Li Cai. Department of Biomedical Engineering, Rutgers University, 599 Taylor Road, Piscataway, NJ
Notch1 signaling is critical for neural development. Its activity is required for the maintenance of neural stem cell properties and neural cell fate determination. Although the downstream pathway regulated by Notch1 is extensively investigated, the molecular mechanism that regulates Notch1 expression is still not fully understood. Here we report the regulatory activity of a recently identified 399 bp cis-element (Notch1CR2, or CR2) in the second intron of Notch1 during embryonic and neonatal development. Using transgenic mouse model, we determined the spatiotemporal activity of CR2 from embryonic day 9.5 to postnatal day 7 in the spinal cord. CR2 was preferentially active in the progenitors of the GABAergic interneurons but not the motoneurons. In summary, our findings may provide new insights into the regulatory mechanism of Notch1 expression in neural development.
Acknowledgements: This work was supported in part by the grants from the National Institute of Health (EY018738) and the New Jersey Commission on Spinal Cord Research (08-3074-SCR-E0; 10-3091-SCR-E-0; CSCR12FEL001). The authors declare that there is no conflict of interests.
8. Disease modeling enabled by large-scale manufacture of robust human iPSC-derived cells
Rachel Llanas, Wen Bo Wang, Blake Anson, Susan DeLaura, Eugenia Jones, David Mann and Vanessa Ott Cellular Dynamics International Madison, WI
Induced pluripotent stem cells (iPSCs) are derived from human somatic cells (e.g. blood, skin) and have the potential to differentiate into any cell type in the human body. In the last 5 years, a large, growing body of literature has emerged demonstrating the use of iPSC-derived cells to recapitulate human disease phenotypes in vitro, commonly referred to as “disease in a dish.” These novel human cell models are being adopted rapidly for disease research, drug discovery, and tissue engineering, offering new avenues for therapeutic intervention. One hurdle to broad application of these models is obtaining cells in sufficient quantities with consistent quality at high purity. Here we present the development of an industrial-scale manufacturing platform for the production of cryopreserved human iPSCs and iPSC-derived cells with high quality and purity. Included among iPSC-derived cells currently available are cardiomyocytes, neurons, astrocytes, hepatocytes, endothelial cells, and hematopoietic progenitor cells. We show how transgene-free, feeder-free iPSCs derived from blood, fibroblasts and LCLs from different donors, both normal and patient, grown in defined media consistently produce high purity cortical neurons. In addition, we show how cardiomyocytes derived from patient iPSCs can be used to elucidate disease mechanisms, including how cardiomyocytes can be used to model cardiac hypertrophy, and will show progress towards the development of a 250 donor iPSC panel for left ventricular hypertrophy. Lastly, we show how access to a consistent supply of pure, high quality iPSC-derived cells is accelerating infectious disease and tissue engineering research. Herein, we postulate that the consistent production of transgene-free, feeder-free iPSC derived cells from a variety of somatic sources and donors, coupled with the production of high quality cryopreserved differentiated cells is needed for the development of disease panels that enable researchers to replicate results across experiments and labs. Robust, reproducible “disease in a dish” research will speed drug discovery and drive a revolution in the movement from bench to bedside.
9. The role of nAChR D398N genetic variant in nicotine addiction
Oni, E. (1), A Halikare(3), A. Toro-Ramos(1), J.C. Moore(2,5,6), M. Swerdel (1), G. Li (3), N. T. Bello(4), J. A. Tischfield (2,5,6), Z.P. Pang(2,3), R.P.Hart (1,2,3) 1 Cell Biology and Neuroscience, Rutgers University, Piscataway, NJ, USA, 2 Human Genetics Institute, Rutgers University, Piscataway, NJ, USA, 3 Child Health Institute, Rutgers-Robert Wood Johnson Medical School, New Brunswick, NJ 4 Department of Animal Sciences, Rutgers University, New Brunswick, NJ, USA 5 Department of Genetics, Rutgers University, Piscataway, NJ, USA, 6 RUCDR Infinite Biologics, Rutgers University, Piscataway, NJ, USA
Many addictive drugs such as nicotine mediate reward and reinforcing mechanisms within the mesolimbic pathway involving midbrain dopaminergic neurons via nicotinic acetylcholine receptors (nAChRs). Previously, genome-wide association analyses (GWAs) identified several nucleotide polymorphism (SNP) associated with increased risk of addictive phenotypes including a SNP encoding a D398N (aspartate to asparagine) variation in the CHRNA5 gene (Saccone et al., 2007; Beirut et al., 2008). Knock-in and expression studies of human CHRNA5 D398N in mice and Xenopus showed increased nicotine consumption, reduction in Ca2+ permeability and increased short-term desensitization to nicotine suggesting a role for the α5 subunit of nAChR in addictive behavior (Changeux, 2010; Fowler et al., 2011; George et al., 2012). Given the evolutionary distance between mice and humans, identifying extracellular factors that affect addiction may correlate poorly, limiting the conclusiveness of these studies. Here we hypothesize that the CHRNA5 D398N SNP causes a modification in nicotine-stimulated Ca2+ influx. Alterations in Ca2+ influx will alter a series of cellular events including neurotransmitter release. Patient-derived induced pluripotent stem cell (iPSC) lines, prepared from cryopreserved lymphocytes, were provided by RUCDR Infinite Biologics in two groups: (1) CHRNA5 D398N, and (2) age, gender-matched unaffected controls. We utilized a neuronal induction culture method (Chambers et al., 2011) to generate mature, nAChR-expressing, dopamine-releasing neurons. These neurons spontaneously fire repetitive action potentials and form synapses as revealed by postsynaptic current responses. Interestingly, with the addition of 3 μM of nicotine, neurons derived from control subjects exhibited less potentiation than D398N genotype. Gene ontology (GO) analysis from total RNA sequencing of 40 day neuronal cultures revealed an increased enrichment of genes associated with neuroactive ligand-receptor interactions and calcium signaling pathways in D398N neurons. Together, these results suggest that the D398N mutation affects Ca2+-signaling in neurons, which may explain the predisposition of subjects carrying this mutation for addictive behavior.
Acknowledgements: NIH R21 DA032984 and NIH Pipeline Grant 5R25GM055145
10. Essential media system for Reprogramming
Rene H. Quintanilla , Jeffrey Fergus, Andrew Fontes, Alexandria Sams, Uma Lakshmipathy Cell Biology and Stem Cell Sciences, Life Technologies, Carlsbad, CA
Essential 8TM media is a simple and cost effective media system for the expansion and maintenance of pluripotent human ESC and iPSC. To extend the use of this system for somatic reprogramming, the basal Essential 6™ media was supplemented with the two growth factors, bFGF and TGFβ, at distinct stages of reprogramming. The optimal timing of growth factor reconstitution to Essential 6™ media was determined for CytoTune® iPSC Sendai and Epi5™Episomal mediated reprogramming of human BJ fibroblasts and human CD34+ blood cells. CytoTune®-mediated reprogramming of BJ fibroblast requires Essential 6™ supplemented with bFGF at early stages for optimal reprogramming efficiencies. In contrast, Essential 8™ media was optimal for reprogramming of CD34+ blood cells both via CytoTune® and Epi5™ reprogramming methods. Further characterization of iPSC derived from BJ fibroblasts confirmed positive pluripotent marker expression, trilineage differentiation potential and normal karyotype. These results and the ability of Essential 8™ media to support fibroblast culture enables a modular feeder-free, defined media system for generation, expansion and maintenance of iPSC.
11. Determination of the Role of Butyrylcholinesterase in Human Neural Stem Cell Differentiation
Tiethof, A. K. Environmental and Occupational Health Sciences Institute and Department of Environmental Medicine, Robert Wood Johnson Medical School and Joint Program in Toxicology, Rutgers University, Piscataway, NJ; Hart, R. P. Rutgers University Department of Cell Biology and Neuroscience and The Human Genetics Institute of New Jersey, Rutgers University, Piscataway, NJ; Richardson, J. R. Environmental and Occupational Health Sciences Institute and Department of Environmental Medicine, Robert Wood Johnson Medical School and Joint Program in Toxicology, Rutgers University, Piscataway, NJ; Moore, J. C. The Human Genetics Institute of New Jersey, RUCDR Infinite Biologics and Rutgers University Department of Genetics, Piscataway, NJ.
Butyrylcholinesterase (BChE), also known as plasma cholinesterase or pseudocholinesterase, is the evolutionary counterpart to acetylcholinesterase (AChE) in the α/β hydrolase fold family of enzymes. AChE functions primarily to enzymatically degrade the neurotransmitter acetylcholine at the neural synapse. BChE also degrades acetylcholine, as well as larger substrates such as butyrylcholine, and other xenobiotics such as cocaine and succinylcholine. Both enzymes appear early in nervous system development prior to cholinergic synapse formation, with BChE expressed prior to AChE. While the role of AChE in the synapse is well-defined, the function of the homologous enzyme BChE is unknown. Since there are no known substrates of BChE present in neural stem cells (NSCs), we hypothesize a non-enzymatic function of BChE in neuronal development. To better understand the role of BChE in human neural development, we used the differentiation of NSCs derived from induced pluripotent stem cells (iPSCs) as a model system. The enzymatic activity of AChE and BChE was characterized, and while activity of both enzymes was detected, only BChE activity increased significantly during early NSC differentiation. Similarly, using quantitative RT-PCR, the expression of both mRNAs was detected, but only BChE was significantly up-regulated during early differentiation of the NSCs. Expression of the transcription factor HES5 is also up-regulated dramatically and significantly early in NSC differentiation. As such, the expression of HES5 mRNA was used as a biomarker to assess the effects of chemical inhibition of AChE and BChE by the pesticide chlorpyrifos on the neuronal differentiation of NSC. Chemical inhibition of AChE and BChE had no effect on the expression of HES5 mRNA on Day 2, 4 or 6 of NSC differentiation. Because there is a hypothesized non-enzymatic role for BChE during neuronal development, shRNA was utilized to knock down expression of BChE to assess the role of BChE on NSC differentiation. Preliminary results indicate BChE knockdown decreases the expression of HES5 mRNA prior to differentiation. These results are consistent with the hypothesis that BChE may have a role in the differentiation of NSCs independent of or in addition to its enzymatic activity.
12. High throughput embryoid body formation and templating for stem cell pluripotency analysis, tumor studies and tissue engineering.
M. L. Tomov, Z. T. Olmsted, J. L. Paluh, Nanobioscience, College of Nanoscale Science and Engineering, SUNY, Albany, NY
Three dimensional (3D) cell aggregates mimic in vivo structures and provide an effective precursor for lineage differentiation. In stem cell biology, embryoid body (EB) formation and analysis provides critical information in evaluating pluripotency and differentiation potential of stem cell lines. However, spontaneously formed EB populations that contain mixed sizes and shapes introduce ‘behavioral variability’ into the experimental design that complicates interpretation of results. We developed lithography-templated arrays (LTA) coated with PDMS (LTA-PDMS) as grids to seed human embryonic stem cells (hESCs) as 2D cell clusters or 3D aggregates. This lithography-based method offers high throughput of embryoid bodies of controllable shape and size and a means to track 3D aggregated cell formation through time-lapse analysis in the transparent template. As observed with mouse ESCs, the size of human embryoid bodies directly affects differentiation potential along ectoderm, endoderm, and mesoderm lineages. By combining EB pre-templating with an additional subsequent post-patterning step, we are able to ensure uniform patterning and settling thus generating results of high statistical accuracy in regard to evaluating and controlling differentiation of these homogeneously formed EBs. We are applying these tools as an in vitro platform for tumor formation in reprogramming studies in cancer research as well as in tissue engineering with 3D-initiated lineage progenitors. Our approach offers an exciting new strategy for studying 3D cell aggregates by enabling better monitoring and control of cell aggregate formation and precise patterning for multiple uses in tissue engineering, tumor cell analysis, and regenerative medicine applications.
Acknowledgements: NYSTEM funded research, C026186.
13. Highly efficient and robust generation of neural stem cells from human pluripotent stem cells using a chemically defined neural induction medium
Yiping Yan, Soojung Shin, Sams Alexandria, David Kuninger, Mohan C. Vemuri Primary & Stem Cell Systems, Life Technologies, 7335 Executive Way, Frederick, MD 21704
Human pluripotent stem cells (hPSCs) including human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) are excellent sources for studies of cell fate specification, disease modeling and drug screening. In order to produce various neural cells from hPSCs, the induction to neural stem cells (NSCs) is the first important step. There is a critical need for robust, streamlined and scalable workflows for generation of NSCs from hPSCs. We have developed a chemically defined neural induction medium which can convert hPSCs into NSCs in one week with 80-90% of efficiency but without the time consuming, laborious processes of embryoid formation and mechanical NSC isolation. Briefly, following hPSC subculture and growth to 10-20% confluence, growth medium was exchanged to neural induction medium and replaced every other day. By day 7 of neural induction, the total cell number typically increased by 30-40 times. To confirm the phenotype of derived cells, immunofluorescent staining for pluripotent marker Oct4 and neural markers including Sox1, Sox2 and Nestin was performed. The results showed that less than 1% of cells expressed Oct4 and 80-90% of cells were positive for Sox1. The percentage of Sox2 and Nestin positive cells exceeded 95%. Further experiments showed that derived cells can be expanded 8-10 fold with each passage. The expanded cells maintained phenotype, exhibited normal karyotype and can be differentiated into neurons, astrocytes and oligodendrocytes. These results suggest that the derived cells from hPSCs are truly NSCs. Microarray analysis showed a 75% overlap in gene expression profiles between human fetal NSCs and those derived using our neural induction medium. In addition, neural induction medium worked for multiple hPSC lines including hESC and hiPSCs generated either by episomal vector or Sendai virus (CytoTune®-iPS Sendai Reprogramming Kit) mediated delivery of reprogramming factors or culture expanded in different media, with similar neural induction efficiency. In conclusion, we demonstrate the development of a new chemically defined neural induction medium enabling efficient generation and expansion of NSCs from hPSCs.
Acknowledgements: This research was supported and conducted by Life Technologies.
14. Regulation of adhesion molecule L1 expression by MeCP2 using a Rett Syndrome patient derived iPS cell line
Yoo, MS.1, OY. Kwon1, C. Carromeu2, A. Muotri2 and M. Schachner1 1W. M. Keck Center for Collaborative Neuroscience, Department of Cell Biology and Neurosciences, Rutgers University, Piscataway, NJ, 2School of Medicine Department, Pediatrics/Rady Children's Hospital Department Cellular & Molecular Medicine, University of California San Diego
Mutations in the Methyl CpG binding protein (MeCP2) gene are the most common cause of Rett Syndrome (RTT). Patients present a wide range of neurobehavioral manifestations, such as muscular weakness, movement disorder and no verbal development. MeCP2 has been described as a transcriptional regulator, found binding to the whole genome and possibly modulating the expression of several nervous system related genes. A gene in close proximity to the MeCP2 gene locus in the X chromosome is the cell adhesion molecule L1, which is involved in neuronal cell migration and survival, axon guidance, myelination and synaptic activity. Although diverse studies have covered the importance of L1 in nervous system development and synaptic plasticity, a relationship between MeCP2 and L1 had yet to be discovered. Using human iPSC derived from a patient with a nonsense mutation in the MeCP2 gene and a related control cell line, we show that the MeCP2 background impacts the expression of L1. L1 was found to be down-regulated in the neural stem cells (NSCs) derived from MeCP2 mutant iPSCs, when compared to the control iPSCs. Neuritogenesis in the mutant NSCs was highly reduced and mutant NSCs attached less firmly on substrate-coated Matrigel at low concentrations when compared to control NSCs. Ectopic expression of L1 led to the rescue of some phenotypes of the mutant NSCs, such as enhanced neuritogenesis and tight adhesion to substrate-coated Matrigel. Finally, using an inducible vector encoding wild type MeCP2, we observed a positive correlation between expression of MeCP2 and L1. Our data indicates that MeCP2 regulates L1 expression directly or indirectly and suggests that deficiency in L1 may account for some of the MeCP2 mutant phenotypes.
Supported by a grant from the New Jersey Commission on Science & Technology
15. Generation of Retinal Ganglion Cell (RGC)-like Cells from Mouse Embryonic Stem Cells by Three-dimensional Culture
Min Zou1, Huijun Luo1,2, and Mengqing Xiang1,* 1 Center for Advanced Biotechnology and Medicine and Department of Pediatrics, Robert Wood Johnson Medical School, Rutgers University, 679 Hoes Lane West, Piscataway, NJ 08854 2 Present address: Department of Biochemistry and Molecular Biology, Mayo Clinic Arizona, Scottsdale, AZ 85259
Retinal ganglion cells (RGCs) of the mammalian eye are highly specialized sensory neurons located in the inner layers of the retina. They receive visual information from photoreceptor cells and transmit them to several regions in the brain. Loss and degeneration of RGCs can lead to damage of the optic nerves, loss of visual field and eventually blindness. In mouse models, transplantation of photoreceptor-like cells derived in vitro from mouse ES cells in both two-dimensional (2D) and three-dimensional (3D) culturing systems has shown promise to treat photoreceptor degeneration conditions. However to date, transplantation of in vitro differentiated RGC-like cells has not been significantly successful due to relatively low induction and integration efficiencies. In this study, we constructed a Brn3b-GFP knock-in mouse ES cell line in which Brn3b, a RGC specific gene, is replaced by GFP. By using this ES cell line and adopting a recently reported 3D culturing system to generate optic cup-like structures from ES cells, we successfully induced the formation of retinal tissue and detected the activation of GFP gene in a subpopulation of cells in this synthetic tissue. By immunostaining, we confirmed that the induced retinal tissue can form stratified structure that expresses distinct retinal cell markers mimicking the in vivo developing embryonic retina. The generation, migration, and marker expression of GFP+ cells in the optic cup-like structure faithfully resemble those of nascent RGCs in the developing mouse embryo. The in vitro induced GFP+, RGC-like cells can be enriched by fluorescence-activated cell sorting (FACS) and used for transplantation with the therapeutic potential for RGC degenerative diseases such as glaucoma.
Acknowledgements: This project is supported by the National Institutes of Health (EY020849 and EY012020 to M.X.)
16. Essential media system for reprogramming
Rene Quintanilla, Jeffrey Fergus, Andrew Fontes, Alexandria Sams, Uma Lakshmipathy
Cell Biology and Stem Cell Sciences, Life Technologies, Carlsbad, CA
Essential 8TM media is a simple and cost effective media system for the expansion and maintenance of pluripotent ESC and iPSC. To extend the use of the system for somatic reprogramming, the basal Essential 6 media was supplemented with the two growth factors bFGF and TGFβ at distinct stages of reprogramming.
The optimal timing of growth factor reconstitution to Essential 6 media was determined for CytoTune and Epi5 mediated reprogramming of human BJ fibroblasts and human CD34+ blood cells. CytoTune-mediated reprogramming of BJ fibroblast required Essential 6 with bFGF at early stages for optimal reprogramming efficiencies. In contrast, Essential 8 media was optimal for reprogramming of CD34+ blood cells both via CytoTune and Epi5 reprogramming methods. Further characterization of iPSC derived from BJ fibroblasts confirmed positive pluripotent marker expression, trilineage differentiation potential and normal karyotype. This and the ability of Essential 8 media to support fibroblast culture enable a modular feeder-free defined media system for generation, expansion and maintenance of iPSC.