SCR 513 Proposal Exam

what is the title for topic 1 (Non-rodent ESCs)?

Reconstructing In Vivo Epiblast Dynamics to Derive Germline-Competent Rabbit Embryonic Stem Cells

What is the main problem of topic 1?

Derivation of germline-competent ESCs is normal in rodents but not in non-rodents, where cells drift into primed states

What is the importance of studying topic 1?

Non-rodents are essential for modeling human disease, enabling large-animal gene transfers, and studying flat-disc embryogenesis without using human embryos

What has been attempted for topic 1?

borrowed mouse culture conditions (2i/LIF); human primed conditions (FGF2/Activin) applied across species

Why is topic 1 still unresolved?

Borrowed media alters bivalent chromatin (H3K4me3/H3K27me3) and cause loss of competence for formative epiblast transition

What is the hypothesis for topic 1?

Reconstructing an in vivo embryo-matched signaling and epigenetic roadmap will enable derivation of rabbit ESCs that stably retain formative transition plasticity and germline competence

What’s the rational behind topic 1’s proposal?

Rabbits share flat-disc embryology with humans while having a 30-day gestation period; matching culture conditions to natural developmental transitions prevents epigenetic drifts and preserves competence

what is the first aim for topic 1?

map the in vivo rabbit epiblast-to-germline transcriptional and chromatin timeline (E3.5 blastocyst to E7.0 early PGC specification

what is the second aim for topic 1?

derive rabbit ESCs under tailored signaling conditions (6iL/E4+TDI) and benchmark molecular accuracy against aim 1’s atlas

what is the third aim for topic 1?

validate functional germline competence via in vitro PGCLC induction and in vivo blastocyst chimera transmission

what is the experimental approach to topic 1 aim 1?

Design: flush rabbit embryos at discrete stages and profile via scRNA-seq, scATAC-seq, and CUT&Tag.

Controls: flushed somatic and primitive endoderm cells (negative)

Expected Results: pinpoints the transient molecular signature of germline competence

what is the experimental approach to topic 1 aim 2?

Design: plate E3.5 rabbit ICMS in multi-inhibitor medium & profile passages P5-P20 by scRNA-seq and CUT&Tag; benchmark against aim 1

Controls: standard 2i/LIF-cultured cells (drift control); fresh ICMs (baseline)

Expected Results: maintains naive markers (OCT4, Nanog, Klf17) without drifting into primed or trophectoderm fates

what is the experimental approach to topic 1 aim 3?

In Vitro Assay: induce PGCLCs with BMP4/8b, SCF, and EGF; quantify DDX4/VASA+, BLIMP1+, and Sox17+ cells via flow cytometry

In Vivo Gold Standard: inject 10-12 GFP+ rESCs into host blastocysts; transfer to psuedopregnant rabbits; confirm genital ridge colonization and breed F0 chimeras to confirm F1 germline transmission

what are the pitfalls of topic 1’s proposal?

cultured cells undergo spontaneous trophectoderm drift due to MEK inhibition; prolonged culture causes global DNA hypomethylation and loss of imprinting

what are the alternative strategies of topic 1’s proposal?

supplement inhibitors to lock out trophectoderm lineage entry; culture under physiological hypoxia or use transient inhibitor exposure

what is the overall impact of topic 1?

resolves barrier of non-rodent naive stem cell capture; provides platform for large-animal genetic engineering and disease modeling

what is the title for topic 2 (Long-Term PGC Expansion)?

Sustaining Mammalian Primordial Germ Cell Self-Renewal via Convergent Wnt and BMP Signaling

what is the main problem of topic 2?

cultured mammalian PGCs fail to expand long-term, either dying or undergoing fate drift into embryonic germ

what is the importance of studying topic 2?

stable mammalian PGC expansion is critical for in vitro gametogenesis, preservation of endangered species, and treating forms of infertility

what are the prior attempts done for topic 2?

culturing PGCs on STO feeders with LIF/bFGF/SCF - promoted proliferation

why is the problem for topic 2 unresolved?

the conditions reprogram PGCs into EG cells that form teratomas and lose lineage-restricted unipotent identity

what is the hypothesis for topic 2?

mimicking physiological WNT3 BMP signaling while sustaining the core repressive network allows long-term self-renewal of mammalian PGCS while suppressing EG/pluripotent fate drift

what is the rationale behind topic 2’s proposal?

mammalian PGCs drift because current media lack the coordinated cues needed to repress pluripotency while sustaining proliferation

what is the first specific aim for topic 2?

establish a chemically defined culture system (WNT3/BMP4/SCF/insulin) that maintains long-term PGC proliferation while preventing pluripotency conversion

what is the second specific aim for topic 2?

verify germline-specific epigenetic reprogramming through global hypomethylation and imprinting erasure across extended serial passages

what is the third specific aim for topic 2?

validate functional gametogenic competence via in vitro gonadal reconstitutions and in vivo transplantation

what is the experimental approach to topic 2 aim 1?

Design: isolate E8.5-E10.5 mouse PGCs engineered with dual reporters (Stella GFP for germline and Nanog-mCherry for EG); culture on feeder-free substrates in base medium supplemented with WNT3a/BMP4/SCF/insulin

Controls: classic EG medium (LIF+bFGF+serum - positive), basal medium with WNT/BMP (negative)

Expected Results: continuous proliferation of Stella-GFP+/Nanog-mCherry cells maintaining PGC markers without colony flattening

what is the experimental approach to topic 2 aim 2?

Design: perform single-cell enzymatic methylation sequencing and PCR for imprinted loci at p2, p10, and p25

Controls: fresh isolated in vivo gonadal PGCs (positive), somatic fibroblasts (methylated control)

Expected Results: demonstrates genome wide DNA hypomethylation and imprint erasure while retaining karyotype

what is the experimental approach to topic 2 aim 3?

In Vitro Gametogenesis: aggregate expanded PGCs with embryonic gonadal somatic cells to generate reconstituted ovaries or testes; culture to induce meiotic entry and gamete maturation

In Vivo Assay: microinject expanded male PGCS into germ-cell depleted tests

Expected Results: generated haploid sperm or MII oocytes capable of IVF and yields viable F1 offspring transmitting donor reporter

what are the pitfalls of topic 2’s proposal?

spontaneous conversion to pluripotent EG cells despite signaling control; cells undergo proliferative arrest or apoptosis after Day 5

what are some alternative strategies for topic 2?

integrate an inducible cassette to enforce continuous transcriptional repression of the pluripotency network; provide membrane-bound SCF using stromal cells to support survival signaling

what is the overall impact of topic 2?

overcomes hurdle of EG fate conversion; establishes the first expandable mammalian PGC culture platform for in vitro gametogenesis, fertility restoration, and epigenetic research

what is the title for topic 3 (Cancer Stem Cells)?

In Vivo Lineage Tracing to Evaluate Phenotypic Plasticity and Stemness Reacquisition in Colorectal Cancer

what is the main problem of topic 3?

the CSC model suggests a rigid unidirectional hierarchy where rare cells drive tumorgenesis; finding show that 28% of single cells form tumors and markers are reversibly expressed (plasticity model)

what is the importance of studying topic 3?

if CSCs are a fixed subpopulation, then targeting them can fix the issue; if they are plastic, non-CSCs will regenerate CSCs causing therapies to fail

what are the prior attempts for topic 3?

surface marker sorting (CD133, CD271, LGR5) paired with limiting dilution xenografts

why is the problem for topic 3 unresolved?

high marker variability, lack of universal definitions, and xenograft assay sensitivity bias

what is the hypothesis for topic 3?

in colorectal cancer, cancer stemness is a dynamic, reversible cellular state governed by microenvironmental signaling, wherein non-CSCs (LGR5-) can reacquire an LGR5+ CSC state and reconstitute long-term tumorigenic capacity

what is the rational behind topic 3’s proposal?

LGR5 marks normal crypt stem cells and CRC-initiating cells; lineage tracing and marker re-analysis allow direct in vivo tracking

what is the first specific aim for topic 3?

isolate LGR5+ and LGR5- populations from patient-derived CRC cells and compare their molecular stemness signatures via sc-RNA seq

what is the second specific aim for topic 3?

functionally evaluate tumor-initiating frequency and serial transplantation capacity of LGR5+ vs LGR5- cells using limiting dilution xenografts in NSG mice

what is the third specific aim for topic 3?

determine whether purified, lineage-marked LGR5- non-CSCs can plastically switch back into functional LGR5+ CSCs in vivo and drive secondary tumor formation

what is the experimental approach to topic 3 aim 1?

Design: dissociate CRC samples; isolate LGR5+/LGR5- cells by FACS; perform scRNA-seq to compare core stemness + pathways

Controls: Normal adjacent mucosa (baseline); isotype gating for FACS purity

Expected Results: LGR5+ cells enrich for stemness signatures but a subset of LGR5- cells retain basal Wnt transcript priming

what is the experimental approach to topic 3 aim 2?

Design: transplant serial dilutions of FACS-sorted LGR5+/LGR5- cells subcutaneously in Matrigel into NSG mice; calculate stem cell frequency via ELDA; perform serial re-transplantations

Controls: unfractioned tumor cells (positive baseline); vehicle-only injections (negative)

Expected Results: LGR5+ cells display significantly higher tumor-initiating frequency and shorter latency; LGR5- cells at higher doses still form tumors

what is the experimental approach to topic 3 aim 3?

Design: transduce LGR5- cells with a reporter; inject labeled LGR5-GFP+ cells into NSG mice; harvest tumor, dissociate, and quantify the presence of LGR5-GFP+ cells via flow cytometry and IF; perform secondary assays

Controls: sorted LGR5+ cells tracked in parallel; re-analysis of sorted population

Expected Results: detection of GFP+/LGR5+ cells in LGR5- tumors proves cellular plasticity and dedifferentiation

what are some pitfalls of topic 3’s proposal?

LGR5- tumor initiation is caused by rare sorted LGR5+ contamination rather than plasticity; subcutaneous mouse microenvironment alters natural CRC growth dynamics

what are some alternative strategies for topic 3?

implement double sorting with post sort purity analysis; perform orthotopic cecal/colonic wall microinjections or utilize patient-derived 3D tumors in defined ECM matrices

what is the overall impact of topic 3?

resolves whether stemness is a rigid cell hierarchy or a plastic state, establishes need for dual-targeting strategies for CSCs and non-CSCs

what is the title for topic 5 (Safe & Precise Genome Editing)?

Targeted Adenine Base Editing in PAX7+ Satellite Cells to Prevent Regenerative Decay in Muscular Dystrophy

what is the main problem for topic 5?

in vivo CRISPR-Cas9 therapies rely on double-strand breaks and episomal AAV delivery to differentiated somatic tissues; DSBs cause large on-target deletions

why is studying topic 5 important?

in DMD, muscle undergoes continuous cycles of breakdown and repair; unless permanently corrected therapeutic benefit will decay

what are th prior attempts of topic 5?

systemic AAV-Cas9 delivered to myonuclei restored reading frames in mdx mice, but blocks re-dosing, prolonged nuclease expression, and fails to efficiently edit satellite cells.

why is topic 5 still unresolved?

bulky editor cargos exceed AAV packaging limits; myonuclear-targeted corrections are diluted out during regeneration

what is the hypothesis for topic 5?

restoring the DMD reading frame in resident muscle stem cells (PAX7+ satellite cells) using transient lipid-nanoparticle-delivered Adenine Base Editor mRNA will achieve durable, lifelong dystrophin expression that persists through repeated degeneration-regeneration cycles

what is the rationale behind topic 5’s proposal?

only edits integrated into the genome of self-renewing PAX7+ satellite cells are inherited by newly regenerated myofibers; transient LNP mRNA deliver confines editor expression to days

what is the first specific aim for topic 5?

compare LNP-ABE mRNA vs AAV9-ABE delivery to quantify editing efficiency separately in isolated PAX7+ satellite cells vs post-mitotic myonuclei in mdx mice

what is the second specific aim for topic 5?

establish on-target base editing precision, quantify genome-wide safety/off-target profiles, and verify restoration of functional, membrane-localized dystrophin complex

what is the third specific aim for topic 5?

determine whether PAX7+ satellite cell editing confers long-term dystrophin persistence through repeated cycles of muscle injury/regeneration and evaluate repeat-dosing safety

what is the experimental approach for aim 1 in topic 5?

Design: treat mdx mice with systemic tail-vein injections of muscle-trophic LNP-ABE8e LNPs targeting AG splice acceptor of DMD exon; benchmark against AAV9-ABE; FACS-sort PAX7+ satellite cells; quantify edit frequencies via sequencing

Controls: AAV injection (pharmacological control); vehicle-only LNPS (negative)

Expected Results: LNP-mRNA will significantly exceed AAV in editing PAX7+ satellite cells

what is the experimental approach for aim 2 in topic 5?

Design: assess on-target amplicons for bystander edits; find off-target sites using GUIDE-seq; perform long-read sequencing across DMD locus to rule out large structural deletions

Controls: wild-type C57BL/6 muscle (positive protein); unedited mdx muscle (negative)

Expected Results: precise AG conversions without DSBs or large deletions

what is the experimental approach for aim 3 in topic 5?

Design: subject treated mdx to 3 rounds of cardiotoxin-induced muscle injury and regeneration; measure dystrophin-positive myofiber density and ex vivo muscle force regeneration; treat with 3 repeat LNP doses and monitor offspring

Controls: myofiber-only edited mice; unchallenged aging cohort

Expected Results: in fiber-edited mice, dystrophin collapses following injury as unedited stem cells replace ribers; in satellite-cell edited mice, dystrophin persists

what are some pitfalls of topic 5’s proposal?

rare satellite cells yield insufficient genomic DNA for deep sequencing; bystander adenines within the ABE editing window produce aberrant splicing/missense mutation

what are some alternative strategies for topic 5’s proposal?

pre-amplify isolated satellite cell DNA using low-cycle nested PCR or ddPCR before sequencing; screen narrowed-window base editor variants to write the exact splice junction without bystander deamination

what is the overall impact of topic 5?

re-defines therapeutic model in genetic degenerative disorders; establishes an LNP-base editing blueprint for durable, lifelong muscular dystrophy treatment

what is the title for topic 6 (Stem Cell-Based Embryo Models)?

Engineering Complete Human Post-Implantation Embryo Models from Chemically Induced Founder Cells

what is the main problem for topic 6?

early human post-implantation development is mostly unknown due to uterine inaccessibility and ethical limits; current stem cell-based embryos suffer low assembly and lack maternal endometrial support

why is studying topic 6 important?

understanding this window is critical to understanding the primary causes of early pregnancy loss, congenital malformations, and defective placental formations

what are the prior attempts of topic 6?

mixing separately primed naive ESCs (epiblast, trophectoderm, hypoblast) or forcing transgenes in mice/humans

why is topic 6 still unresolved?

separately primed cells cause developmental asynchrony and fail to coordinate spatial symmetry; lack receptive maternal interface

what is the hypothesis for topic 6?

transiently inducing human iPSCs into a single “embryo founder-like” state with multilineage plasticity, combined with receptive 3D endometrial organoid co-culture, will improve structural self-organization, with symmetry breaking, and post-implantation development

what is the rationale behind topic 6’s proposal?

in vivo early blastomeres generate all embryonic and extraembryonic tissues from a single coordinated founder state; providing an endometrial niche provides physiological cues needed for survival and implantation

what is the first specific aim for topic 6?

induce and validate a human embryo “founder-like” state from naive iPSCs capable of forming all three blastocyst lineages

what is the second specific aim for topic 6?

assemble founder-derived embryo models and assess structural accuracy

what is the third specific aim for topic 6?

co-culture models with hormone-receptive endometrial organoids to evaluate implantation and early post-implantation survival

what is the experimental approach for aim 1 in topic 6?

Design: treat naive human iPSCs with transient small molecules modulating Wnt, TGF-beta, and ERK signaling to induce co-expression of early lineage specifiers; quantify using scRNA-seq and flow cytometry

Controls: untreated naive iPSCs (negative baseline)

Expected Results: transient small-molecule exposure reprograms naive iPSCs into a viable uncommitted founder state without dominant lineage restriction

what is the experimental approach for aim 2 in topic 6?

Design: aggregate founder-like cells in microwells and centrifuge to allow self-organization; evaluate models across these criteria: lineage composition, spatial architecture, and developmental progression

Controls: multicellular embryoid bodies (disorganized control); separately mixed lineage models

Expected Results: founder cells self-organize in 3D into integrated models containing an outer trophoblast shell, posterior primitive streak elongation, and 3-germ layer gastrulation

what is the experimental approach for aim 3 in topic 6?

Design: establish hormone-responsive, 3D endometrial organoids containing estrogen and progesterone; co-culture embryo models with organoids to model implantation stages

Controls: models cultured without organoids; models cultured with non-receptive organoids

Expected Results: live-cell imaging of invasion, hCG secretion assays; presistence of yolk sac and epiblast confirm increased embryo survival and implantation compared to organoid-free controls

what are some pitfalls of topic 6’s proposal?

founder cells fail to specify one of the three lineages, spontaneous A-P symmetry breaking fails or occurs in few models

what are some alternative strategies for topic 6’s proposal?

supplement transient pulses of BMP4 or use a modular fallback; apply a localized microfluidic gradient of Wnt/BMP to establish a single signaling pole

what is the overall impact of topic 6?

establishes a scalable, single-source platform to build complete human embryo models, provides an ethical system to investigate early pregnancy failure

what is the title for topic 7 (MSC therapies)?

Enhancing Allogeneic MSC Efficacy and Homing in Acute GVHD via Endothelial Progenitor Cell Crosstalk

what is the main problem of topic 7?

MSCs possess potent immunomodulatory and regenerative capacity but clinical translation is limited by batch-to-batch heterogeneity, unpredictable outcomes, and trial failures

why is studying topic 7 important?

steroid-refractory Graft-vs-Host Disease occurs in 30-50% of allogeneic bone marrow transplants and carries high mortality

what are the prior attempts of topic 7?

trials use standard surface markers and plastic adherence to release cell batches, assuming phenotype guarantees therapeutic function

why is topic 7 still unresolved?

surface markers do not reflect in vivo immunosuppressive potency; donor variability is high, infused cells rapidly undergo apoptosis or fail to home to inflamed tissues

what is the hypothesis of topic 7?

standardizing xeno-free expansion, selecting batches via a quantitative functional potency matrix, and pre-conditioning MSCs with endothelial progenitor cells (EPCs) will eliminate functional variability and ensure durable suppression of alloreactive T cells in GVHD

what is the rationale behind topic 7’s proposal?

MSCs therapeutic benefit is drive by paracrine factor secretion and immune re-education; functional in vitro use and vascular crosstalk with EPCs enhance cell survival and tissue homing

what is the first specific aim for topic 7?

establish a standardized, xeno-free manufacturing and quality-control protocol to eliminate batch-to-batch MSC heterogeneity and prevent replicative deterioration

what is the second specific aim for topic 7?

develop a multi-parametric mixed lymphocyte reaction potency matrix to quantitatively screen and release MSC batches based on functional immunosuppresion

what is the third specific aim for topic 7?

evaluate whether EPC pre-conditioning enhances MSC survival, migration, and therapeutic efficacy in a humanized mouse model of acute GVHD

what is the experimental approach for aim 1 in topic 7?

Design: isolate hBM-MSCs from qualified healthy donors and expand in low-glucose DMEM supplemented with pooled human platelet lysate under physiological hypoxia; use flow cytometry and western blot

Controls: adult BM-MSCs cultured in standard FBS to late passage (senescence control)

Expected Results: yields uniform MSC batches through passgae 4 that maintain tri-lineage differentiation potential and show minimal cellular senescence

what is the experimental approach for aim 2 in topic 7?

Design: perform co-culture MLRs by incubating candidate MSC batches with allogeneic peripheral blood mononuclear cells stimulated with alloantigens

Controls: stimulated PBMCs without MSCs (max inflammation); resting PBMCs (baseline)

Expected Results: potent MSC batches demonstrate dose-dependent immunosuppresion in MLRs, reducing activated T cell proliferation by > 50%, expanding regulatory T cells, and establishing high anti-inflammatory secretome ratio

what is the experimental approach for aim 3 in topic 7?

Design: establish acute GVHD in NSG mice by sub-lethal irradiation followed by injection of human PBMCs; infused either unconditioned MSCs, pre-conditioned MSCs, or vehical controol

Controls: healthy untransplanted mice; GVHD mice treated with standard steroids

Expected Results: EPC-preconditioned MSCs exhibit superior in vivo survival and enhanced vascular homing to target organs in humanized acute GVHD mice

what are some pitfalls of topic 7’s proposal?

primary bone marrow donors exhibit unavoidable biological variance in potency; donor cells are rapidly cleared from circulation before reaching target tissues

what are some alternative strategies for topic 7’s proposal?

transition to human umbilical cord blood MSCs to establish an infinite, rejuvenation-stable mater cell bank; deliver MSC-derived extracellular vesicles rather than whole cells to lower risk of pulmonary trapping

what is the overall impact of topic 7?

shifts the MSC field from a surface-marker-driven industry to a standardized, potency therapeutic discipline; establishes a release-testing blueprint to reliably treat refractory GVHD

what is the title for topic 8 (Alzheimer’s Disease Cell Therapy):

Dual-Engineered Neural Progenitors and Exosome Priming for Amyloid Clearance and Hippocampal Synatic Repair

what is the main problem of topic 8?

alzheimer’s disease involves progressive loss of hippocampal neurons and synaptic circuits driven by amyloid-beta plaques, neuroinflammation, and loss of neurotrophic support

why is studying topic 8 important?

over 55 million individuals live with dementia globally; current treatments only provide modest symptom slowing and fail to restore lost synapses or replace dying neurons

what are the prior attempts of topic 8?

transplanting unmodified neural stem cells provided paracrine BDNF support without clearing plaques; neprilysin-overexpressing NSCs degraded amyloid-beta without direct synaptic repair