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What Stem Cell & Lineage Differentiation got wrong, from 53 theses

Summary written by a model from the records below: The records document recurring experimental and procedural challenges encountered during stem cell maintenance, directed lineage differentiation, and tissue modeling. Research efforts frequently failed due to off-target lineage commitment, culture detachment, spontaneous loss of pluripotency, genetic perturbation toxicity, and protocol inefficiency. These records come from graduate theses, 2021 to 2026, each labeled with its degree where the repository states it, and each links to its thesis. They were extracted by language models reading the full text, so treat each as a lead to read, not a verdict.

53
theses
17
institutions
56
failure records
6
failure modes

Plus 10 problems the authors left open, listed at the end.

Failure modes, by theses reporting them

Select a mode to jump to its records.

Directed differentiation protocols produced off-target lineages and heterogeneous cell populations
13
13 theses · 10 institutions · 13 records
Protocols and cell lines failed to achieve efficient differentiation into functional target cells
11
11 theses · 5 institutions · 12 records
Cultured cells suffered from substrate detachment, excessive aggregation, and shear-induced death
9
9 theses · 7 institutions · 10 records
Pluripotent stem cells and progenitors underwent spontaneous differentiation or loss of potency
8
8 theses · 6 institutions · 8 records
Targeted genetic perturbations caused cell cycle arrest, cell competition, or differentiation failure
7
7 theses · 6 institutions · 7 records
Co-culture systems and engineered niches failed to support proper differentiation and integration
5
5 theses · 4 institutions · 6 records

Directed differentiation protocols produced off-target lineages and heterogeneous cell populations

13 theses · 10 institutions

Modulating differentiation with small molecules or mechanical cues frequently promoted off-target somatic lineages instead of the desired cell types. Cultures also suffered from extensive batch-to-batch variation, low target purity, and species-specific differentiation biases.

Tried and failed

histone deacetylase inhibitor treatment applied to stem cell differentiation to germline identity. Outcome: worse than baseline. Reason: impaired differentiation dose-dependently and promoted somatic lineage gene activation instead

EFFICIENT GENERATION OF PRIMORDIAL GERM CELLS AND CRISPR PERTURBATION SCREENS IDENTIFY EPIGENETIC REGULATORS IMPACTING PRIMORDIAL GERM CELL DEVELOPMENT · Cornell · PhD

Tried and failed

extended small molecule Wnt agonist exposure applied to stem cell differentiation in stirred bioreactors. Outcome: worse than baseline. Reason: prolonged activation of signaling pathway disrupted target lineage specification during dynamic culture

Biomanufacturing of Kidney Organoids, Perfusable Proximal Tubules, and Kidney Tissues · Harvard · PhD

Tried and failed

directed lineage differentiation of heparan sulfate-deficient cells applied to human pluripotent stem cells. Reason: EXT1 knockout prevents mesendoderm and primitive streak lineage commitment despite preserving ectoderm differentiation

Roles for cell surface glycans in guiding human pluripotent stem cell fate · MIT · PhD

Tried and failed

Wnt signaling attenuation to rescue differentiation applied to chromatin remodeler knockout stem cells. Reason: rescued aberrant off-target gene expression but failed to restore target lineage marker expression

Investigating the role of the chromatin remodeller CHD3 during human cranial neural crest cell specification · Imperial · PhD

Lost to a baseline

Modified small molecule differentiation protocols (early CHIR, altered BMP/PUR/RA timing) were poorer than the standard baseline Koehler protocol, yielding significantly more off-target cells.

Generation of human inner ear organoids from pluripotent stem cells and an organ-on-chip platform · Boston University · Master's

Considered and rejected

Considered and rejected: Small-molecule-based dual-SMAD inhibition differentiation protocols, rejected due to differentiation heterogeneity, inconsistency, and lengthy neural progenitor stages compared to NGN2 overexpression.

EPIGENETIC REGULATION IN AN IPSC-NEURON MODEL OF FAMILIAL ALZHEIMER’S DISEASE · Penn · PhD

Considered and rejected

Considered and rejected: Rejected directed differentiation via smNPCs for disease modeling due to lower purity (10-35% vs 50-80%) and high batch-to-batch variability

Neurodevelopmental Deficits and Aberrant Neuron-Neuron Interactions in iPSC-derived Psychiatric Disease Models · Tübingen · PhD

Tried and failed

early retinoic acid receptor inhibition during differentiation applied to stem cell cardiomyocyte differentiation. Outcome: worse than baseline. Reason: expanded cardiac mesoderm but severely blocked downstream differentiation into functional cardiomyocytes

Use of Cell- and Gene-Based Therapies for Providing Cardiac Biological Pacing · Georgia Tech · PhD

Tried and failed

alternative transcript isoform overexpression applied to inducing lineage differentiation in cell lines. Outcome: worse than baseline. Reason: the alternative isoform repressed cellular proliferation and differentiation instead of promoting lineage commitment

lncRNA CRNDE204 regulates erythropoiesis and myelopoiesis by binding to PUS1 · Harvard · Master's

Considered and rejected

Considered and rejected: Rejected undirected differentiation for microcircuit modeling in favor of directed lentiviral transcription factor induction because undirected protocols yield variable, heterogeneous mixtures and take longer.

Neurodevelopmental Alterations in Idiopathic and Isogenic iPSC-derived Psychiatric Disease Models · Tübingen · PhD

Considered and rejected

Considered and rejected: Rejected human iPSC-derived liver organoids due to long differentiation protocols (~1 month), loss of expansion capacity once differentiated, uncontrolled batch-to-batch variation across complex cell types, and restriction to modeling early embryonic development.

Dissecting the role of biliary epithelial cells during non-alcoholic fatty liver disease progression · EPFL · PhD

Tried and failed

increasing hydrogel matrix stiffness and elasticity applied to hematopoietic stem cell T-lineage differentiation. Reason: stiff elastic matrices suppressed T-cell commitment and biased differentiation toward myeloid lineage

Amplifying mechanotransduction in human T-cell development to enhance immunotherapies · Harvard · PhD

Tried and failed

cyclic mechanical stretch and pharmacological cues applied to mesenchymal stem cell differentiation. Outcome: no signal. Reason: most cells co-expressed pericyte markers instead of achieving pure endothelial lineage differentiation

Mechanobiological rejuvenation of mesenchymal stem cell therapeutics · UT Austin · PhD

Protocols and cell lines failed to achieve efficient differentiation into functional target cells

11 theses · 5 institutions

Differentiation strategies and single transcription factor inductions frequently failed to generate functional target cells or induce spontaneous contractility. Furthermore, lines derived from different donors, sexes, or patient cohorts showed substantial resistance or failed quality control thresholds.

Tried and failed

single transcription factor overexpression for directed differentiation applied to pluripotent stem cell gamete differentiation. Outcome: no signal. Reason: Individual transcription factors were insufficient on their own to drive cell fate conversion

Characterization of transcription factor function during human gametogenesis via directed differentiation · Harvard · PhD

Tried and failed

directed stem cell differentiation applied to male induced pluripotent stem cells. Outcome: no signal. Reason: insufficient responsiveness to differentiation cues for female tissue-specific lineages compared to female cell lines

Towards a Non-Invasive Model for Women’s Uterine Health: From Transcriptional Programs to Tissue Assembly · Harvard · PhD

Lost to a baseline

Original definitive endoderm differentiation protocol performed worse on IPF patient-derived hiPSCs compared to healthy REBLPAT cells.

In vitro modelling of respiratory infections in idiopathic pulmonary fibrosis (IPF) using human induced pluripotent stem cell (hiPSCs)-derived alveolar epithelial type II cells · Nottingham · Doctorate

Lost to a baseline

3 iPSC cell lines (WTSIi096, WTSIi051, WTSIi017) excluded from differentiation to MGLs due to failing flow cytometry quality control thresholds (< 80% CD11b+/CD14+/CD45+).

Phenotypic characterisation of sex differences in myeloid cells · Imperial · PhD

Considered and rejected

Considered and rejected: STEMdiff Hematopoietic commercial monolayer differentiation kit was rejected in favor of spin EB differentiation because 3 of 12 runs failed to yield suspension cells and progenitor colony-forming efficiency was inconsistent.

Understanding the role of HOXA9 in haematopoiesis · Nottingham · PhD

Considered and rejected

Considered and rejected: Did not use eWAT SVF for in vitro differentiation assays because eWAT cells have little to no differentiation capacity in vitro.

Role of GPS2 in the regulation and maintenance of adipose tissue physiology and function under metabolic stress and obesity · Boston University · PhD

Tried and failed

Wnt-pathway modulation for directed cardiomyocyte differentiation applied to rabbit induced pluripotent stem cells. Outcome: no signal. Reason: Wnt inhibitor protocols yielded negligible cardiomyocyte efficiency and no spontaneous beating in rabbit iPSCs

Feasibility and efficacy of pluripotent stem cell derived cardiomyocytes and endothelial cells for cardiac regeneration · Imperial · Doctorate

Considered and rejected

Considered and rejected: Direct hanging drop embryoid body cardiac differentiation protocol was rejected for rabbit iPSCs due to feeder cell dependency and low efficiency

Feasibility and efficacy of pluripotent stem cell derived cardiomyocytes and endothelial cells for cardiac regeneration · Imperial · Doctorate

Considered and rejected

Considered and rejected: Rejected P19CL6 embryonic carcinoma cells as the primary cellular differentiation model due to high experimental inconsistency, low beating efficiency, and malignancy compared to mESCs.

Transcriptomic analysis of the role of Hdac4 in cardiac differentiation of mouse embryonic stem cells · Nottingham · PhD

Considered and rejected

Considered and rejected: Rejected using non-isogenic disease and control iPSC lines for modeling monogenic neurodegenerative disease due to differentiation potential variability and donor background masking disease pathways.

Characterization of TDP-43-related de novo proteins in ALS/FTD · Oxford · PhD

Considered and rejected

Considered and rejected: Abandoned multi-stage iPSC-derived microglia differentiation in favour of direct PBMC monocyte differentiation due to extensive culture time (45 days) and poor differentiation reproducibility.

Design and Evaluation of a Human Microglia Model: Drug Delivery and the Inhibitory Impact of PPARβ Agonist on Inflammation · Nottingham · PhD

Considered and rejected

Considered and rejected: Rejected the cytokine cocktail protocol for iPSC hematopoietic differentiation due to adherent cell trapping and low yield.

Investigating the SUMOylation and transcriptional regulation of Nurr1 and the splice variant Nurr-1a · Nottingham · PhD

Cultured cells suffered from substrate detachment, excessive aggregation, and shear-induced death

9 theses · 7 institutions

Cells undergoing differentiation frequently detached from substrates as sheets or formed concatenated aggregates in suspension culture. Attempts to dissociate or replate differentiated cells generated high shear forces that caused extensive clumping and cell death.

Tried and failed

immediate cell replating post-lineage specification applied to stem cell differentiation into hepatocytes. Reason: poor cell attachment to the substrate immediately following definitive endoderm induction

Improving differentiation and maturation of hESCs-derived hepatocytes for biomedical applications · Imperial · PhD

Tried and failed

3D suspension embryoid body differentiation applied to human pluripotent stem cell differentiation. Reason: Neurospheres aggregated into concatenated masses causing poor attachment, low yield, and delayed lineage specification

Investigating the role of sall4 during human cranial neural crest cells specification · Imperial · PhD

Lost to a baseline

ALMS1-iPSCs showed lower attachment, higher post-ROCK-inhibitor-withdrawal cell death, and lower differentiation stability than the standard H9 hESC baseline (reaching only 60% vs 80% confluency in 3 days).

UNDERSTANDING CARDIOMYOCYTE DIFFERENTIATION FROM PLURIPOTENT STEM CELLS · Johns Hopkins · Master's

Considered and rejected

Considered and rejected: Rejected direct differentiation of iPSCs in situ on 3D scaffolds due to cell loss risks during differentiation, opting to seed pre-differentiated cardiomyocytes.

Building 3D architectures for cardiomyocytes · Nottingham · PhD

Considered and rejected

Considered and rejected: Rejected shifting breast-derived broiler satellite cells into serum-restricted differentiation medium (2% HS) because it induced massive cell loss and poor differentiation; maintained in growth medium instead

Characterisation of the anabolic effects of leucine on primary chicken muscle cells and murine C2C12 muscle cells · Nottingham · PhD

Tried and failed

bulk differentiation followed by replating applied to neural stem cell cultures. Reason: dissociating mature differentiated cells caused clumping requiring high shear forces that induced cell death

A Novel In Vitro Model to Investigate Chemotherapy-Related Cognitive Impairment · Harvard · Master's

Tried and failed

growth factor withdrawal after cell attachment applied to neural stem cell differentiation in microplates. Reason: growth factor depletion after cell adherence led to excessive cell overconfluency

A Novel In Vitro Model to Investigate Chemotherapy-Related Cognitive Impairment · Harvard · Master's

Lost to a baseline

3D differentiation in all STH formulations resulted in significantly lower O4+ oligodendrocytes (STH-2: 13.63%, STH-3: 17.30%) compared to 2D laminin culture baseline (23.24%).

Towards Improved Myelination: Engineering Bioinstructive Scaffolds for Schwann Cell Differentiation and Shear-Thinning Hydrogels for Oligodendrocyte Progenitor Therapy · University at Buffalo · PhD

Tried and failed

osteogenic differentiation culture applied to bone marrow mesenchymal stem cells. Outcome: unstable. Reason: persistent cell proliferation caused sheet detachment before nodule formation

Adaptation Of Maternal Skeletal Mechano-Responsiveness, Osteocyte Microenvironment, And Bone Marrow Adipocytes In Response To Reproduction And Lactation · Penn · PhD

Considered and rejected

Considered and rejected: Culturing osteogenic differentiation without dexamethasone (OB-D) rejected due to consistent cell sheet detachment and lack of mineralization

Differenzierung von osteogenen Vorläuferzellen aus induzierten pluripotenten Stammzellen · Tübingen · PhD

Pluripotent stem cells and progenitors underwent spontaneous differentiation or loss of potency

8 theses · 6 institutions

Attempts to maintain naive pluripotency or expand stem cell populations resulted in spontaneous lineage differentiation and loss of stem cell markers. Overconfluence, serum exposure, basal media choices, and genetic modifications also accelerated unwanted differentiation or rapid dedifferentiation.

Tried and failed

transcription factor knockout for cell state transition applied to primed pluripotency reprogramming. Reason: loss of factor caused severe growth arrest and spontaneous differentiation instead of naïve state transition

The role of SOCS3 in nuclear reprogramming and naïve pluripotency network integration · Cambridge · PhD

Tried and failed

chemical inhibitor cocktail primed-to-naive stem cell conversion applied to human embryonic stem cells. Outcome: unstable. Reason: cells entered transitional state with spontaneous differentiation and loss of pluripotency markers

Exploring the function of NLRP7 in regulating human maternal imprinting · Imperial · PhD

Tried and failed

genetic ablation of target signaling complex applied to human embryonic stem cell maintenance. Outcome: unstable. Reason: loss of target complex induced spontaneous lineage differentiation in chemically defined media during extended passaging

Investigating the role of RICTOR/mTORC2 in regulating self-renewal and differentiation of human embryonic stem cells · Imperial · PhD

Lost to a baseline

Peg13 DMR DEL2 clonal ESC line failed differentiation into induced neurons (iNs) due to loss of pluripotency, precluding gene expression analysis in this line.

Investigating the cis-regulatory mechanisms underlying neuronal imprinted expression · Harvard · PhD

Considered and rejected

Considered and rejected: Rejected culturing iPSCs to massive overconfluence to reduce size heterogeneity because overconfluence causes spontaneous differentiation and loss of stem cell potency.

Microfluidic Cell Processing for Personalized and Regenerative Medicine · Georgia Tech · PhD

Considered and rejected

Considered and rejected: Rejected fetal calf serum supplementation during culturing because it drives unwanted differentiation and diverges gene expression from patient primary tissue.

Therapeutic targeting of PRMT5 and mutant ACVR1 in paediatric glioma · Oxford · PhD

Considered and rejected

Considered and rejected: Rejected using RPMI-based custom expansion media for erythroid progenitor induction studies because basal media trended toward premature differentiation compared to SFEM II.

Investigating the cellular response to folate depletion · Harvard · PhD

Considered and rejected

Considered and rejected: Rejected using primary hepatocyte monoculture due to rapid dedifferentiation and loss of physiological function within 24-72 hours.

Establishment and characterization of advanced hepatic cell culture models for the early assessment and mechanistic understanding of drug-induced liver injury · Tübingen · PhD

Targeted genetic perturbations caused cell cycle arrest, cell competition, or differentiation failure

7 theses · 6 institutions

Knockouts and CRISPR perturbations designed to direct cell specification frequently arrested cell growth or caused widespread apoptosis. Perturbed cells failed to advance past early progenitor stages or were completely outcompeted by wild-type cells.

Tried and failed

transcription factor knockout for terminal cell differentiation applied to pluripotent stem cell derived endocrine progenitors. Reason: cells failed to progress past progenitor stage into mature hormone-producing lineages

Loss Of Tbx3 Enhances Pancreatic Progenitor Generation From Human Pluripotent Stem Cells · Penn · PhD

Tried and failed

constitutive gene knockout before differentiation applied to hematopoietic stem cell differentiation. Outcome: worse than baseline. Reason: knockout impaired progenitor proliferation and severely reduced final differentiated cell yield

Enhancing human NK cell development: from UCB-CD34+ cells to functional NK cells · Imperial · PhD

Considered and rejected

Considered and rejected: Rejected targeting IRF4 knockout to block plasma cell differentiation because IRF4KO caused substantial proliferative defects causing cells to be outcompeted by wild-type cells

Modulating antigen-specific humoral immunity with non-differentiating B cells · University of Washington · PhD

Tried and failed

single-gene knockout for cell fate reprogramming applied to neuronal subtype specification. Reason: Perturbations caused incomplete differentiation or cell death rather than transdifferentiation to alternative subtypes

Development of bipolar interneuron subtypes within the retinal space-time continuum · Harvard · PhD

Tried and failed

CRISPRi knockdown of endogenous p53 applied to lineage reprogramming to motor neurons. Outcome: worse than baseline. Reason: p53 knockdown decreased target cell yield compared to control reprogramming conditions

Molecular Mechanisms Defining and Driving Receptivity in Conversion of Fibroblasts to Motor Neurons · MIT · PhD

Considered and rejected

Considered and rejected: Rejected interferon-knockout mouse models because lack of interferon disrupts normal baseline neural progenitor proliferation and differentiation

Effects of Zika virus on neural precursor cell types and microencephaly in a model of direct embryonic murine brain infection · Boston University · PhD

Tried and failed

multiplex CRISPR gene knockout applied to hematopoietic progenitor cell differentiation. Reason: gene targeting halted cell proliferation and differentiation at early to mid stages

CRISPR/Cas9 targeting of single genes in human blood cell progenitors to study Natural Killer cell development · Imperial · PhD

Co-culture systems and engineered niches failed to support proper differentiation and integration

5 theses · 4 institutions

Introducing adult endothelial cells, alternative stromal feeders, or preformed beds failed to enhance tissue differentiation and prevented matrix integration. Additionally, pre-differentiated cells reverted to proliferative states or failed to recreate primary marrow and myotube phenotypes.

Tried and failed

co-culturing primary adult endothelial cells during differentiation applied to stem cell derived organoid development. Outcome: worse than baseline. Reason: inhibited target tissue differentiation and failed to improve vascular integration

Vascularized, Immune-Infiltrated, and Perfusable Kidney Organoid-on-Chip Models · Harvard · PhD

Considered and rejected

Considered and rejected: Rejected continuing with in vitro spike-in transgenic control models because they failed to recapitulate the viability, differentiation, and polarization of thawed post-alloSCT marrow cells

Graft-versus-leukaemia antigen discovery utilising the bone marrow T cell repertoire in patients with acute myeloid leukaemia · Oxford · PhD

Tried and failed

sequential pre-differentiation prior to co-culture seeding applied to multi-cell-type in vitro co-culture. Outcome: unstable. Reason: pre-differentiated myocytes reverted to mitotic proliferation and overgrown post-mitotic neurons

In vitro microfluidic platform for co-culture of myocytes with mouse embryonic stem cell-derived interneurons and motor neurons · UT Austin · PhD

Tried and failed

hydrogel substrate conditioning of stem cell secretome applied to in vitro myogenic differentiation assay. Outcome: worse than baseline. Reason: hydrogel-stimulated conditioned media failed to improve myotube formation over tissue culture plastic controls

Culture systems for controlling mesenchymal stromal cell, protein, and cell-secreted protein release · Georgia Tech · PhD

Tried and failed

alternative stromal feeder co-culture applied to hematopoietic stem cell differentiation modeling. Outcome: no signal. Reason: WJ-MSCs failed to recapitulate the lymphoid progenitor phenotype observed with bone marrow stromal cells

Characterizing the role of ETV6 in lymphoid development and B-cell acute lymphoblastic leukemia predisposition · Harvard · Master's

Tried and failed

seeding aggregates onto preformed stromal cell beds applied to organoid co-culture in hydrogels. Outcome: worse than baseline. Reason: preformed cell bed prevented matrix integration and hindered differentiation

Vascularized, Immune-Infiltrated, and Perfusable Kidney Organoid-on-Chip Models · Harvard · PhD

Left open by the authors

Problems the authors named and did not get to.

Left open

Test whether extracellular vesicles derived from differentiated cells act as signaling cues to induce embryonic stem cell differentiation. Blocker: Requires wet-lab cell culture, EV isolation, and stem cell differentiation assays

EXTRACELLULAR VESICLES IN STEM CELL BIOLOGY · Cornell · PhD

Left open

Differentiate TBK1 mutant stem cells into microglia, astrocytes, and macrophages to assess non-cell-autonomous neuroinflammation mechanisms. Blocker: Requires a wet lab, stem cell lines, and cell culture/differentiation protocols

TBK1 deficiency leads to TDP-43 pathology driven by endo-lysosomal dysfunction in human-induced motor neurons · Harvard · PhD

Left open

Pattern dual opposing gradients of Shh and BMP4 on nanoneedle substrates to direct mouse embryonic stem cell differentiation across the dorsal-ventral axis. Blocker: Requires a wet lab, microfabrication facilities (DRIE/RIE), biomaterial deposition, inkjet printing, and stem cell culture

Engineering in vitro cellular models through spatial patterning of morphogens · Imperial · Doctorate

Left open

Develop culture protocols and small-molecule steering regimens to extend gastruloid viability beyond 168 hours and differentiate them into thymus or parathyroid lineages. Blocker: Requires a wet-lab cell culture environment, mouse embryonic stem cell lines, and specialized biochemical reagents

In vitro endoderm emergence and self-organisation in the absence of extraembryonic tissues and embryonic architecture · EPFL · PhD

Left open

Differentiate human embryonic stem cells into neurons and glial cells to validate SETX-mediated R-loop, ncRNA, and nucleolar aggregate dynamics in vitro. Blocker: Requires wet lab facilities, hESC cell lines, differentiation protocols, and molecular biology assays

Senataxin deficiency disrupts proteostasis through non-coding RNA expression and nucleolar protein aggregation · UT Austin · PhD

Left open

Determine the mechanistic link between TBX3, EMT, and cytoskeletal dynamics during pancreatic endocrine cell delamination and late beta-cell differentiation. Blocker: Requires a wet lab with human pluripotent stem cell culture, differentiation protocols, and molecular biology assays.

Loss Of Tbx3 Enhances Pancreatic Progenitor Generation From Human Pluripotent Stem Cells · Penn · PhD

Left open

Model and test whether preDC differentiation into cDC1 and cDC2 occurs exclusively in bone marrow or in peripheral circulation. Blocker: Requires experimental in vivo lineage tracing or tissue-specific kinetic data to distinguish marrow versus peripheral differentiation sites

Mathematical modelling of human immune cell lineages in vivo · Imperial · PhD

Left open

Isolate and culture Gli1+ periodontal crestal stem cells in vitro to evaluate their proliferation, self-renewal, and multilineage differentiation capacity. Blocker: Requires wet lab access, transgenic mouse models, cell culture facilities, and differentiation assays

Gli1+ Stem Cells Contribute to the Regeneration of Periodontal Tissues · Harvard · Master's

Left open

Perform combinatorial genetic perturbations to identify epistatic interactions during stem cell-derived islet differentiation. Blocker: Requires wet lab CRISPR perturbation experiments in stem cell-derived 3D cultures

Controlling Cell Fate During Directed Differentiation of Human Beta Cells · Harvard · PhD

Left open

Co-culture healthy iPSC-CMs with FRDA-iPSC-CMs to evaluate potential rescue of the FRDA cellular phenotype. Blocker: Requires wet lab cell culture facilities and stem cell differentiation capabilities

Developing induced pluripotent stem cell derived cardiomyocytes as a model to study Friedreich's ataxia · Imperial · PhD

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