Estudiantes actuales con becas activas
Jackson Rogow NIH NRSA F31 Fellowship for a project entitled “Regulation of Presynaptic Gene
Transcripts During
Synaptogenesis” (Sponsor, Peri Kurshan, Neuroscience)
Elliot Kim NIH NRSA F30 Fellowship for a project entitled “Effects of locomotion on visual
inter-areal processing”
(Sponsor, Adam Kohn, Neuroscience)
Daniella Miller NIH NRSA F30 Fellowship for a project entitled “Investigation of Chromatin
Modifiers to Elucidate the
Phenotypic Variability of Congenital Heart Disease in Patients with 22q11.2 Deletion Syndrome” (Sponsor, Bernice
Morrow,
Genetics)
Brett Bell NIH NRSA F30 Fellowship for a project entitled “Anti-Complement Immunotherapy for
Pancreatic Cancer”
(Sponsor, Chandan Guha, Pathology)
Helen Jung NIH NRSA F30 Fellowship for a project entitled
"Strategies for next-generation flavivirus vaccine development "
(Sponsor, Jon Lai, Biochemistry)
Riana Lo Bu NIH NRSA F31 Fellowship for a project entitled
" Dissecting GWAS Identified Risk Variants in Parkinson's Disease –
Functional Role of GPNMB in the Pathogenesis of PD " (Sponsor, Frank
Soldner, Neuroscience)
Vanessa Ruiz NIH NRSA F31 Fellowship for a project entitled
"Characterizing subsets of HIV-infected and uninfected CD14+CD16+ monocytes
that contribute to neuropathogenesis" (Sponsor, Joan Berman, Pathology)
Jessie Larios Valencia NIH NRSA F31 Fellowship for a
project entitled "The Role of Dedifferentiation in Basal like Breast Cancer"
(Sponsor, Wenjun Guo, Cell Biology)
Erik Guillen NIH NRSA F31 Fellowship for a project entitled “Impact of T cell receptor
signaling on memory CD8+ T cell
stemness” (Sponsor, Gregoire Lauvau, Microbiology & Immunology)
Eric Sosa NIH NRSA F31 Fellowship for a project entitled
"Defining the gene regulatory roles of non-coding variants in the
pathogenesis of autism" (Sponsor, John Greally, Genetics)
Tram Nguyen NIH NRSA F30 Fellowship for a project entitled
"Reward Function in Adolescents with Depression and Cannabis Use"
(co-Sponsors, Vilma Gabbay and Benjamin Ely, PCI-Neuroscience &
Psychiatry)
Gabriel Bedard NIH NRSA F30 Fellowship for a project
entitled "Rational design of anti-cancer therapeutics harnessing the
synthetic lethality of methionine metabolism and arginine
methyltransferases" (Sponsor, Vern Schramm, Biochemistry)
Matanel Yheskel NIH NRSA F31 Fellowship for a project
entitled "Epigenetic and transcriptional consequences of Intellectual
Disability-associated mutations in the histone lysine demethylase KDM5"
(Sponsor, Julie Secombe, Genetics)
Andrea Bae NIH NRSA F30 Fellowship for a project entitled
"Role of brain oscillations in midbrain and forebrain networks supporting
stimulus selection in the sound localization pathway of barn owls" (Sponsor,
Jose Luis Pena, Neuroscience)
Jacob Stauber NIH NRSA F30 Fellowship for a project
entitled "Understanding stem-cell evolution dynamics of donor clonal
hematopoiesis in allogeneic hematopoietic cell transplantation at a
single-cell level" (co-Sponsors, John Greally and Ulrich Steidl, Genetics
and Cell Biology)
Ian MacArthur NIH NRSA F30 Fellowship for a project
entitled "Epigenetic regulation of neural stem cell biology by Tet DNA
dioxygenases" (Sponsor, Meelad Dawlaty, Genetics)
Leti Nunez NIH NRSA F31 Fellowship for a project entitled
"Determining the effect of RNA binding protein phosphorylation on mRNA fate"
(Sponsor, Robert Singer, Anatomy and Structural Biology)
Chris Nishimura NIH NRSA F30 Fellowship for a project
entitled "Mechanistic Dissection and Therapeutic Targeting of B7x in Cancer"
(Sponsor, XingXing Zang, Microbiology & Immunology)
Ryan Graff NIH NRSA F30 Fellowship for a project entitled
"Platelet PI3Kβ regulation of metastasis" (Sponsor, Jonathan Backer and Anne
Bresnick, Molecular Pharmacology)
Daniel Borger NIH NRSA F30 Fellowship for a project
entitled "Developing a novel ex vivo platform to support hematopoietic cells
and characterize the stem cell niche" (Sponsor, Paul Frenette, Cell Biology)
Estudiantes actuales con becas completadas
NHenrietta Bains NIH NRSA F31 Fellowship for a project entitled "How does mTOR sense lipid in
vivo" (Sponsor, Rajat
Singh, Developmental & Molecular Biology)
Julio Flores NIH NRSA F31 Fellowship for a project entitled "Epigenetic regulation of stem
cells and development by the
DNA dioxygenase Te2" (Sponsor, Meelad Dawlaty, Genetics)
Michelle Gulfo NIH NRSA F31 Fellowship for a project entitled "Assessing dopaminergic
modulation of an associative
circuit within the dentate gyrus" (Sponsor, Pablo Castillo, Neuroscience)
Resúmenes de proyectos recientes
Eric Sosa - ABSTRACT: In this Predoctoral Fellowship
proposal, I will be trained for a future as a physician-scientist with my
own independent research program at the interface of genomics, computational
biology, and neuroscience. During my MD/PhD training, I will be co-mentored
by two physician-scientists, Drs. John Greally (Medical Genomics) and Pablo
Castillo (Neurology), addressing a question that is timely with the imminent
widespread use of whole genome sequencing (WGS) in clinical diagnostics –
how do we interpret variants in the noncoding majority of the human genome
when a patient presents with a medical problem? I will focus on autism, as a
condition that represents a substantial proportion of patients seen by
medical genetics services, for which there is extensive WGS information from
thousands of families. Despite this wealth of research sequencing, only a
small minority of individuals with autism receive a positive outcome of
diagnostic exome or WGS. I propose that the currently limited diagnostic
success rates are mostly due to our inability to interpret pathogenic
variants in the non-coding majority of the genome of these patients. By
improving our insights into non-coding variants, we will be able to offer
diagnostic information to many more families seeking answers than currently.
My strategy is to focus on de novo variants (DNVs) in offspring with autism
born to unaffected parents. My hypothesis is that DNVs can be pathogenic
when they occur in the cis-regulatory regions of cell types mediating
autism. The project is therefore based on a computational genomics
foundation, using WGS and DNV calls from large datasets from thousands of
families who have a member with autism. In my preliminary data, I show that
DNVs in individuals with autism are enriched at cis-regulatory loci in glial
and neuronal cells in particular, and at genes known to be causative for
autism. In my project, I will test these associations more rigorously, and
will define a high-confidence set of DNVs for functional testing. Two types
of functional testing will be performed. One will test whether the DNVs
alter molecular genomic properties, including chromatin accessibility and
gene expression. The second will test the physiological properties of the
cells. To generate the appropriate cells for testing, I plan to use induced
pluripotent stem cells (iPSCs) that are in vitro differentiated to GABAergic
neurons and astrocytes. By performing CRISPR-mediated genomic editing in the
iPSCs, I can generate cells with the candidate pathogenic DNVs, and test
whether they have effects on cellular properties like dendritogenesis,
synaptogenesis, and electrophysiology, increasing the confidence that these
DNVs have pathogenic effects. I will have the privilege of getting training
in sophisticated computational, stem cell and neuroscience techniques, under
the guidance of two leaders in their fields, as part of a comprehensive
training plan that will equip me to become the independent
physician-scientist I aspire to be in my career.
Tram Nguyen - ABSTRACT: Adolescent depression and cannabis
use are concurrent major public health concerns, and this comorbidity has
been associated with long-term cognitive and behavioral consequences.
However, there has been sparse research in this area, as most neuroimaging
studies in adolescent depression exclude cannabis users. We seek to address
this gap based on converging evidence that: 1) Reward dysfunction
contributes to maintenance and progression of depression in adolescents; 2)
Reward dysfunction is a heterogenous construct involving deficits in diverse
cognitive processes, including reward anticipation, attainment, and
prediction error; 3) These deficits entail distinct neural mechanisms that
can be studied by functional magnetic resonance imaging (fMRI); 4) The major
psychoactive agent in cannabis, Δ9-tetrahydrocannabinol (THC), exerts its
effects through modulation of the cortico-striatal reward system; and 5)
Cannabis use may result in temporary relief of mood and anxiety symptoms
while inducing potentially deleterious long-term neural reward alterations.
We documented that anhedonia–a core symptom of depression reflecting reward
deficits–was associated with worse depression outcomes, including chronicity
and suicidality, in adolescents. Similarly, using resting-state fMRI, we
found that altered striatal intrinsic functional connectivity (iFC) with the
prefrontal cortex was associated with anhedonia severity in depressed
adolescents. Further, using the reward flanker task (RFT), we found that
adolescents with diverse mood and anxiety symptoms showed weaker striatal
activation during reward anticipation than healthy controls, while stronger
activation in the anterior cingulate cortex predicted worse higher levels of
anhedonia at two-year follow-up. We therefore propose a 2×2 cross-sectional
study to test the overall hypothesis that comorbid cannabis use in
adolescent depression is associated with more severe neural reward deficits
and clinical symptoms. Participants will comprise 30 depressed cannabis
users, 30 depressed cannabis non-users, 30 non-depressed cannabis users, and
30 healthy controls, all ages 14-18 (Tanner stage ≥ 4) and
psychotropic-medication-free. Using resting-state and RFT task fMRI, we
predict that comorbid cannabis use and depression will be associated with
more pronounced deficits in reward anticipation (Aim 1) and alterations in
cortico-striatal iFC (Aim 2) than depression alone. Further analyses
(Exploratory Aims) will assess the relationships between cannabis use
frequency and activation during reward anticipation, cortico-striatal iFC,
and symptom severity. Public Health Relevance: This proposal aims to address
a critical and growing public health concern that to date has received
minimal research attention while providing a rigorous framework for
predoctoral training in both clinical research and advanced neuroimaging
techniques. Findings from this work will expand the limited understanding
regarding the impact of cannabis on reward dysfunction in youth, with
potential to inform both clinical practice and government policies on
cannabis use.
Gabriel Bedard - ABSTRACT: Methionine adenosyltransferase 2
alpha (MAT2A) and protein arginine methyltransferase 5 (PRMT5) are cancer
targets that are synthetically lethal with MTAP deletions and have several
drug candidates in clinical trials targeting MTAP-/- cancers. MTAP is
deleted in ~15% of human cancers and encodes the metabolic enzyme
5’-methylthioadenosine phosphorylase, the sole enzyme in humans responsible
for recycling of methylthioadenosine (MTA) to methionine. MAT2A synthesizes
S-adenosyl methionine (SAM), the methyl donor substrate for
methyltransferase reactions. PRMT5 utilizes SAM as a substrate and is
inhibited by MTA, and MTAP-/- cells in culture demonstrate elevated MTA
levels. In vivo observations of glioblastoma tumors suggest however, that
MTAP-/- does not always lead to increased tumoral MTA levels due to MTA
efflux into matrix MTAP-competent cells. Additionally, MTAP deletions are a
rare (~2%) occurrence in colorectal cancers (CRCs), precluding MAT2A and
PRMT5 inhibitors’ use for most CRCs. The Schramm laboratory has previously
solved the transition state (TS) structure of MTAP and synthesized a potent
small molecule inhibitor methylthio-DADMe-immucillin-A (MTDIA) that
recapitulates the in vitro effects of MTA accumulation within tissues. MTDIA
has been shown to inhibit tumor growth in several cancer models, including
CRC, and is linked to a decrease in PRMT5 activity through elevation of MTA
levels. We propose that MTDIA be used in combination with MAT2A inhibitor
AG-270, currently in Phase I clinical trials, to harness their synthetic
lethality by targeting PRMT5. We will test the safety, target engagement,
and anti-cancer efficacy of MTDIA in combination with AG-270 in ApcMin/+ and
CRC patient-derived xenograft (PDX) mice. To determine mechanisms of
anti-cancer effects, we will probe the upstream and downstream effects
related to PRMT5 activity. We will perform tumor metabolomic quantification
of relevant metabolites and histone and protein-arginine methylation
characterization using immunohistochemistry and proteomic techniques. We
will also profile the gene expression changes using single-cell RNA
sequencing to determine how combination therapy alters tumor architecture
and growth. Finally, we will solve the transition state structure of PRMT5
with the goal of laying the foundations for development of novel transition
state analogue inhibitors. This work will expand upon the use of MAT2A and
PRMT5 inhibitors beyond the ~15% of MTAP-deleted cancers and provide avenues
for MTDIA to be used in clinical trials.
Ian MacArthur - ABSTRACT: The ten-eleven translocation
family (TET1/2/3) of enzymes are epigenetic regulators of gene expression
that are highly expressed in neural stem cells (NSCs) and during mammalian
nervous system development. TET enzymes are dioxygenases that promote active
and passive DNA demethylation by converting 5-methylcytosine (5mC) into
5-hydroxymethycytosine (5hmC) and higher-order oxidized derivatives. In
addition to its role as a demethylation intermediate, 5hmC can function as a
stable epigenetic mark and is highly enriched and dynamic in the developing
nervous system. TET enzymes and 5hmC dysregulation have been implicated in
human neurodevelopmental syndromes, intellectual disability, craniofacial
abnormalities, and neurodegeneration. These observations suggest a critical
role for TET enzymes in the developing nervous system and has led to
interest in their roles in the biology of NSCs. However, the functions of
TET enzymes in NSCs and neurodevelopment remain poorly understood.
Preliminary data from our lab demonstrates that Tet tripleknockout NSCs
(T123–/–) derived from embryonic stem cells exhibit severe defects in
self-renewal, multipotency, and expression of neurodevelopmental genes. We
therefore hypothesize that TET enzymes have essential functions in
epigenetic regulation of gene expression programs critical for NSC
maintenance and multipotency and in embryonic neurodevelopment. To test this
hypothesis, we have derived embryonic forebrain NSC lines containing floxed
alleles of Tet1/2/3 and a tamoxifen-inducible Cre recombinase transgene
expressed from the constitutive Rosa26 locus (T123F/F; +/R26-CreER) for
conditional, combined deletion of all three Tet genes. We have also
established a colony of Tet1/2/3 triple-floxed mice expressing a
tamoxifen-inducible Cre recombinase transgene under control of the
neural-specific Nestin promoter (T123F/F; +/Nestin-CreERT2). Using these
models, we will (1) define the role of TET enzymes in the maintenance and
multipotency of NSCs , (2) establish the requirement of TETs in embryonic
neurodevelopment, and (3) identify TET-mediated epigenetic and
transcriptional regulatory mechanisms in NSCs. Findings from these studies
will define novel roles played by TET enzymes in NSC biology and
neurodevelopment, provide insights into how dysregulation of TETs contribute
to human neurodevelopmental disorders, and identify novel targets for
therapy.
Chris Nishimura - ABSTRACT: Immune checkpoints are proteins
that regulate the body’s immune system via inhibition of immune cells. In
diseases such as cancer, these same pathways may be commandeered in order to
inappropriately inhibit immune responses. One approach to overcome this
excessive inhibition and restore normal immune function is to physically
block these inhibitory proteins using monoclonal antibodies (mAbs), a
strategy known as immune checkpoint blockade. Our lab discovered the protein
B7x (VTCN1/B7S1/B7-H4), an immune checkpoint whose regulation and mechanism
of action are still being elucidated. B7x is expressed on a wide variety of
cancers and is associated with poor clinical outcomes. It has been shown to
inhibit effector T cell functions such as cytokine production and
proliferation, and promotes T cell exhaustion; it has also been associated
with tumor infiltration of immunosuppressive cell populations such as
myeloid derived suppressor cells (MDSCs). Intriguingly, it is not typically
co-expressed with the well-studied immune checkpoint PD-L1, and blocking B7x
using anti-B7x mAbs improves anti-tumor responses and survival in mouse
models. This suggests that B7x holds a non-overlapping but key role in
cancer immune evasion. For these reasons I hypothesize that B7x mediates
immune evasion through inhibiting effector cell functions while
simultaneously promoting immunosuppressive cells and is therefore a
promising target for cancer immunotherapy. Thus, we propose the following
two aims: 1) Examine the mechanisms that regulate B7x expression and its
role on MDSCs; and 2) Develop and characterize a new anti-B7x immunotherapy.
In aim 1 I will examine the how hypoxia and other tumor
microenvironment-associated cytokines regulate B7x expression. In addition,
I will explore how B7x affects the immunosuppressive MDSC cell population
generation and survival. In aim 2 I will test the anti-tumor efficacy of
newly generated anti-B7x mAb-based immune checkpoint blockade in vivo using
metastasis and spontaneous models of lung cancer. We will also characterize
how this new immunotherapy alters T cell phenotypes using flow cytometry and
single-cell RNA sequencing. Finally, we will explore if combining our
anti-B7x mAb with anti-TIM3 and anti-PD1 mAbs confers superior anti-tumor
efficacy than anti-B7x monotherapy in vivo. Together, these studies will
allow us to broadly explore the role of B7x in tumorigenesis and therapeutic
potential of anti-B7x immunotherapy, and combined with a personalized
training plan and supportive research environment at Einstein, cultivate the
skills required of a physician-scientist.
Ryan Graff - ABSTRACT: Metastasis is the primary cause of
morbidity and mortality among patients with solid tumors. Platelets interact
with tumor cells upon their entry into the vasculature and promote the
metastatic spread of these cells by several mechanisms. Circulating tumor
cells (CTCs) directly bind to and activate platelets resulting in the
formation of platelet-fibrin complexes that envelope circulating tumor cells
and protect them from immune clearance. Adhesion to tumor cells also induces
the release of platelet cytokines and other soluble factors that promote
epithelial-mesenchymal transition in the tumor cells. Finally, platelets
promote the adhesion of CTCs to the endothelium, assisting in their
extravasation at distant metastatic sites. Platelet activation, adhesion,
and in vivo thrombus formation require the activity of the Class IA PI
3-kinase PI3K. This isoform of PI3K produces the majority of PI(3,4,5)P3 in
platelets, suggesting that PI3K has a unique role in classical platelet
activation. However, the role of PI3K in platelet-mediated cancer
metastasis has yet to be defined. We propose that PI3K is required for
platelet activation upon interaction with tumor cells. Aim 1 will test the
role of platelet PI3K in platelet-stimulated tumor cell Matrigel invasion,
transendothelial migration, epithelial-mesenchymal transition, cell
stemness, NFB activation, and platelet TGF secretion. We will also use
RNAseq and antibody arrays to more broadly examine the role of PI3K in
platelet chemokine/cytokine release and in transcriptional responses by
tumor cells. We will use both platelets from mice expressing mutant PI3K
and human platelets pre-treated with the irreversible pan-PI3K inhibitor
wortmannin. Aim 2 will directly evaluate the role of platelet PI3K in
cancer metastasis in wild type and whole-animal or platelet-specific mutant
PI3K mice. Murine mammary carcinoma cells will be injected intravenously to
analyze in vivo tumor cell-platelet complex formation as well as tumor cell
interactions with macrophages and other leukocytes. We will also test the
role of PI3K in tumor cell-induced thrombocytopenia and experimental
metastasis. Isoform selective PI3K inhibitors were originally developed to
be used as anti-thrombotic agents, as they prevent thrombotic occlusion of
injured arteries without increasing bleeding. More recently these inhibitors
have also been explored as anti-cancer agents in the treatment of
PTEN-deficient cancers. This proposal seeks to understand the potential for
a broader application of these agents in cancer patients to inhibit platelet
activity and prevent metastasis in a variety of cancer types.
Henrietta Bains - ABSTRACT: Alterations in lipid metabolism
determine metabolic disease and mortality in the aging population. Despite
our understanding of regulation of lipid metabolism, how organisms sense
lipid remains unknown. It is conceivable that sensing of lipid will inform
downstream decisions taken by the cell that modulate metabolism,
proteostasis, stress response, and growth—each of which are dysregulated
with age. The mechanistic target of rapamycin (mTOR), is a serine/threonine
kinase and amino acid sensor, that drives growth and proliferation. More
recently, mTOR in cultured cells has been shown to be activated by
cholesterol and phosphatidic acid (PA) in absence of amino acids. Whether
mTOR senses lipid in whole organisms is unclear. mTOR exists as two major
complexes—mTORC1 and mTORC2. Activation of mTORC1 occurs at the lysosomal
surface in presence of amino acids and requires key regulatory proteins that
stimulate its activity. By contrast, mTORC2 responds to growth factors to
regulate cytoskeletal organization. Hyperactivation of mTORC1 (hereafter,
mTOR for simplicity) drives aging and age-related diseases in part by
disrupting autophagy and promoting growth. Indeed, suppressing mTOR has been
shown to increase lifespan in multiple organisms. However, how mTORC1 is
hyperactivated with age remains unknown. It has been shown that there are
quantitative and qualitative changes in membrane lipids with age including
changes in lysosomal membrane lipids—the major site of mTOR activation.
Whether changes in lysosomal membrane lipids are mechanistically linked to
mTOR hyperactivation remain unknown. Our preliminary data show that
subjecting mice to an oral gavage of corn oil causes activation of mTOR and
its translocation to distinct cholesterol-rich microdomains (CRMs)/lipid
rafts in lysosome membranes. Our preliminary data also show that
immunoprecipitating mTOR from lysosome membranes from livers of oil-gavaged
mice reveal its binding to diacylglycerol. These data suggest that mTOR is a
sensor of diacylglycerol, a membrane lipid. Since mTOR senses nutrients at
lysosome membranes, I hypothesize that mTOR senses lipid at lysosomal
membranes, and that age-related changes in lysosomal membrane lipid
composition lead to mTOR hyperactivation. To test our hypothesis, we present
the following specific aims: Aim 1: To determine how mTOR senses lipid at
the lysosome surface. In Aim 1, diverse approaches will be used to
characterize lipid-driven mTOR activation at lysosome membranes. By pulling
down mTOR from lysosome membranes for lipidomic and proteomic analyses, I
will identify lipid species that bind to mTOR and its interacting partners.
I will use an siRNA screen in vitro to silence each of the interacting
partners, which will identify novel regulators of lipid-driven mTOR
signaling. Aim 2: To determine the mechanism of mTOR hyperactivation with
age. In Aim 2, I will characterize the changes in lipid composition of
lysosome membrane CRMs and expansion of lysosome CRMs with age. I will
determine whether alterations in membrane lipid composition with age
correlate with increased mTOR activity. I will then determine whether
inactivating the synthesis of specific membrane lipids, e.g., PA and DG, by
shRNAs against relevant biosynthetic enzymes in liver will dampen
age-related mTOR hyperactivation. I will also determine whether targeting
key interacting partners of mTOR in liver will dampen age-related
hyperactivation of mTOR signaling and reverse deleterious mTOR-dependent
outcomes, i.e., blockage of autophagy and proteostasis failure.
Julio Flores- ABSTRACT: The Ten-eleven translocation
(Tet1/2/3) family of enzymes are epigenetic regulators of gene expression
important for stem cell biology and embryonic development. Tet enzymes are
dioxygenases that promote DNA demethylation by converting 5-methylcytosine
(5mC) into 5-hydroxymethycytosine (5hmC) and higher oxidized derivatives. In
addition to this enzymatic activity, Tet enzymes can bind chromatin
modifying complexes, to regulate genes in a presumably catalytic-independent
manner. Tet2 is a key member of this family. It is highly expressed in
embryonic stem cells (ESCs) and controls gene expression programs necessary
for stem cell lineage specification. Tet2 is also frequently mutated in
hematological malignancies and has been implicated in neurodegenerative
diseases. While the catalytic functions of Tet2 have been well studied, its
non-catalytic roles remain poorly defined. In this proposal, we seek to
establish the significance of the catalytic dependent and independent
functions of Tet2 in ESC gene regulation and lineage commitment. We
hypothesize that Tet2, in addition to regulating genes through its DNA
demethylase activity, can also modulate genes in a non-catalytic fashion by
recruiting histone modifiers to the chromatin, and this dual mode of gene
regulation is essential for proper ESC differentiation along the neural and
hematopoietic lineages. To test this hypothesis, I have generated Tet2
catalytic mutant (Tet2m/m) and knock-out (Tet2–/–) ESCs, which I will use as
a platform to: (1) identify the catalytic and non-catalytic direct target
genes of Tet2 in ESCs by integrating changes in gene expression with Tet2
genomic occupancy, (2) establish Tet2-mediated activating and repressing
mechanisms of gene regulation involving interactions with histone modifiers
OGT and HDAC2, and finally (3) define the biological significance of Tet2
enzymatic and non-enzymatic functions in ESC differentiation and lineage
commitment along the neural and hematopoietic lineages. Findings from these
experiments will elucidate novel epigenetic mechanisms of gene regulation in
ESCs involving Tet2 catalytic and non-catalytic functions. They will enhance
our understanding of stem cell biology and development and can have
implications in hematological malignancies where Tet2 is affected.
Daniel Borger- ABSTRACT: Current culture methods reduce the
ability of hematopoietic stem cells (HSCs) to successfully engraft in a
host. Emerging gene editing technologies such as CRISPR/Cas9 require time in
culture to allow for the correction of disease-causing alleles. There is
therefore a need to develop new methods of culturing HSCs. Coculture of HSCs
with bone marrow niche cells such as mesenchymal stem cells (MSCs) is one
possible solution to problems in HSC culture, as these cells provide factors
that support HSCs in vivo. However, MSCs cannot be maintained in culture for
extended periods of time and fairly rapidly lose expression of niche
factors. Through a screen of candidate transcription factors, our lab
identified 5 factors that when transduced together restore niche factor
expression and allow for prolonged culture. These factors are Kruppel-like
factor 7 (Klf7), Osteoclast stimulating factor
(Ostf1), X-box binding protein (XBP1),
Interferon regulatory factor 3 (Irf3), and Irf7, which we
collectively dubbed the KOXII factors. KOXII-transduced MSCs were able to
expand both murine and human funtional HSCs to a much greater extent than
mock-transduced MSCs. These cells therefore may be useful in expanding HSCs
ex vivo for gene correction. However, there are regulatory barriers to the
application of murine cells in human therapeutics. The work proposed here
will in part focus on the development and characterization of
KOXII-transduced human MSCs. After generating these cells, I will determine
if the KOXII factors affect expression of niche factors in human MSCs. I
will also use flow cytometry and stem cell xenotransplantation to determine
if KOXII-transduced MSCs are more effective at driving HSC expansion than
unmodified MSCs. Finally, using CRISPR/Cas9-based gene editing of HSCs
derived from patients with sickle cell disease as a model, I will determine
if coculture of patient cells with KOXII-transduced MSCs can improve the
efficiency of gene editing or increase the yield of properly edited cells
over current standard HSC culture methods. In parallel, I will use murine
KOXII-transduced MSCs to more closely examine niche signalling by MSCs. As
these cells can be cultured in relatively large numbers, they are ideal for
proteomic studies. In collaboration with the lab of Jeroen Krijgsveld, I
will examine the secretome of these cells in order to identify proteins
whose secretion is upregulated by the KOXII factors. Using both in vitro and
in vivo assays, I will evaluate the effect of these factors on HSC
maintenance and proliferation, with the aim of identifying secreted proteins
with previously unappreciated roles in MSC-HSC niche interactions.