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Open research questions in Pluripotent Stem Cells Research

75 unresolved questions extracted from the limitations and future-work sections of 306 Pluripotent Stem Cells Research papers in our library. Each links back to the study that raised it.

What the literature leaves open

  • Pierre Gönczy (EPFL) provided valuable comments on centriole elimination mechanisms through published work [22,23]; the central experiment of centriole elimination + reprogramming to totipotency has not been reported in the literature.

    Eliminate, Reprogram, and Rebuild · 2026 · DOI

  • Alternative promoter (AP) usage profoundly expands transcriptomic and proteomic diversity, yet the regulatory principles governing promoter choice remain poorly understood.

    A Promoter Competition Hub Orchestrates Runx1 Alternative Promoter Usage during Skeletal Muscle Stem Cell Activation · 2026 · DOI

  • Adult neural stem cells (NSCs) are transcriptionally heterogeneous, yet the relationship between molecular heterogeneities and individual NSC trajectories remains unclear.

    Linking transcriptome to cell behavior in real time uncovers molecular fate-asymmetry and a quiescence cycle among adult neural stem cells · 2026 · DOI

  • During mouse development, yolk sac (YS)-derived macrophages populate the aorta-gonad-mesonephros (AGM) region at the time of HSC emergence, but the mechanisms by which they support ex vivo hematopoietic stem and progenitor cell (HSPC) generation remain poorly defined.

    The Aorta-Gonad-Mesonephros niche shapes the functions of yolk sac-derived macrophages involved in hematopoietic stem and progenitor cell generation ex vivo · 2026 · DOI

  • Hematopoietic stem cell (HSC) longevity critically depends on maintaining a deep dormant state, yet the molecular mechanisms that preserve this rare and functionally essential population remain poorly understood.

    USP7 maintains hematopoietic stem cell dormancy and function by stabilizing HMGA2 · 2026 · DOI

  • The regulation of human hematopoietic stem cell (HSC) function within its native bone marrow microenvironment remains poorly understood due to the limitations of existing humanized models.

    Deploying cross-species bone marrow niches to elucidate human hematopoietic stem cell regulation in vivo · 2026 · DOI

  • Our findings demonstrate the relevance of studying the dynamics upon differentiation rather than just one fixed timepoint and offer a valuable basis for further investigation of non-coding variation in CNCC-related disorders.

    Time-resolved inference of gene regulatory networks underlying human cranial neural crest development suggests novel risk genes for orofacial clefting. · 2026 · DOI

  • Despite extensive work in model organisms, a time-resolved, multi-omics perspective of GRNs controlling CNCC differentiation in a human system is still lacking.

    Time-resolved inference of gene regulatory networks underlying human cranial neural crest development suggests novel risk genes for orofacial clefting. · 2026 · DOI

  • Restoration of Fthl17c expression rescued TET activity, reduced DNA methylation, and reinstated pluripotency associated gene expression, whereas extracellular Fe 2 ⁺ supplementation alone was insufficient.

    MSI1-FTHL17C-iron circuit couples metabolic and epigenetic control of pluripotency in mouse embryonic stem cells · 2026 · DOI

  • Furthermore, a key limitation of CRISPRi was demonstrated using the ATF5-NUP62 locus, wherein CRISPRi could co-repress genes with overlapping promoter regions.

    Comparative benchmarking of CRISPRi and CasRx in standardized pluripotent stem cell platforms reveals context-dependent knockdown performance · 2026 · DOI

  • Implementation of this protocol requires availability of both Cas9 knockin and Cdh5-Cre mouse strains. Establishing a homozygous line for both Cas9 active and Cdh5-Cre alleles requires a pro- longed, multi-generational breeding scheme. Although our protocol offers EC-specific modeling and relatively rapid generation of stable knockout mice that holds significant potential for long- term phenotypic studies, particularly in modeling vascular homeostasis and disease progression, we cannot rule out the possibilities of toxicity of conventional Cre expression, off-target effects from the chosen sgRNAs, and mosaicism with incomplete gene editing in all target cells due to absence of distinct phenotypes and lack of longitudinal studies. Besides, female and male mice were included in both experimental groups and there were no sex-specific differences in gene edit- ing outcomes. Further investigations with higher N numbers for both sexes are warranted to solidify this observation. TROUBLESHOOTING Problem 1 Variability in knockout efficiency between individual mice, e.g., CRISPR mosaicism (Step 3; very faint PCR band observed in the Cas9 mutant positive samples in Figure 1B) and limitation of ordinary genotyping PCR detection (Figures 1A and 1B).

    Protocol to generate endothelial cell-specific knockout mouse models using Cas9/Cdh5-Cre mice coupled with sgRNA · 2026 · DOI

  • Organoids are set to continue delivering important advances in fundamental biology and biomedical applications. Progress in the field will come from balancing developing more complex models while maintaining the advantages of simpler systems to study diverse biological processes, including otherwise inaccessible human developmental mechanisms, as well as for disease modelling and drug testing. Although the earlier organoid models did not recapitulate the complete native microenvironment, this limitation is also a strength, offering a reductionist approach to study epithelial systems in Review article isolation. For example, combining organoids with genome engineering enables bottom–up reconstruction of complex diseases such as cancer, providing nuanced insight into their underlying genetic complexities. This approach makes it possible to generate larger cohorts of raredisease organoid models using genome engineering or by establishing living biobanks. Furthermore, genome engineering enables the generation of reporters and lineage tracing constructs, and we anticipate its central role in dissecting functional dynamics and molecular mechanisms to address increasingly complex biological questions. The repertoire of organoids is expanding beyond mouse and human, and we predict that ‘first-generation’ models from additional species will provide new knowledge through evolutionary comparisons, species-specific physiology and, as demonstrated by a recent bat organoid collection264, studies of viral infection mechanisms and tropism. Increasing organoid complexity enables investigations of interactions between different cell types and more holistic studies of human development and disease. Hence, there are clear advantages and a growing need to further increase the complexity of current models. Although the culture medium supplies the necessary factors for differentiation of specific cell types, the outcome depends on the intrinsic potency of the organoids’ stem/progenitor cell population. The starting material is, therefore, of utmost importance. For adult organs in which the stem/progenitor cells are unipotent, we envision that assembloids and OoC approaches will help increase epithelial heterogeneity. Examples of the assembly approach include the generation of liver buds from induced PSCs265. A recent example shows how assembloids of adult hepatocytes, cholangiocytes and mesenchymal cells can recapitulate liver periportal architecture and allow modelling of cholestatic injury and biliary fibrosis86,266. Although we are moving towards more complex co-cultures containing non-epithelial cell types, much work remains before endogenous epithelial complexity can be fully recapitulated. As the assembloids approach does not require expensive specialized equipment or extensive technical training, it is relatively easily accessible, and we therefore expect this approach to be used more frequently in the future. Co-cultures will be essential to investigate interactions between epithelial cells and their microenvironment, such as mesenchymal, neuronal and immune cells, but also to study interactions between different epithelial tissues, such as placental organoids and decidua267. Overall, organoid co-culture technology remains at an early stage, yet we foresee progress driven by assembloid approaches and the synergy between organoid and engineering approaches. Recently, this potential was elegantly demonstrated by combining genome engineering, optogenetics, OoC methodologies and live imaging to study colon cancer initiation over several weeks268. The integration of organoid technology with artificial intelligence is another area wherein we predict strong synergy. The latest artificial intelligence models predict the structure of complexes, including proteins, nucleic acids and small molecules269, and peptide binding specificity270, and they help generate unprecedented volumes of hypotheses based on morphological and functional data. Translating these predictions into real-world clinical benefit requires rigorous testing and validation. Organoids will be essential to this process by providing platforms for functional testing and generating human in vitro datasets at scale, which are key for training artificial intelligence models to enhance their accuracy in predicting human physiology.

    New developments and applications of human organoids · 2026 · DOI

  • The gastruloids were cultured for a maximum of five days post-induction; extension of culture duration combined with proteomic profiling would reveal whether gastruloid development plateaus or recapitulates later stages of gastrulation and axial mesoderm patterning.

    The proteomic landscape and temporal dynamics of human and mouse gastruloid development · 2026 · DOI

  • Mouse gastruloids and human RA-gastruloids used different induction protocols, media formulations, and sampling timepoints. Direct proteomic comparison between synchronized mouse and human gastruloid development under standardized culture conditions would clarify species-specific versus conserved proteomic signatures.

    The proteomic landscape and temporal dynamics of human and mouse gastruloid development · 2026 · DOI

  • The proteomic landscape was generated from bulk gastruloid tissue; single-cell or spatial proteomics approaches would be required to map cell-type-specific protein expression patterns across the different germ layers (epiblast, mesoderm, endoderm) within gastruloids.

    The proteomic landscape and temporal dynamics of human and mouse gastruloid development · 2026 · DOI

  • Genetic perturbations using CRISPR-Cas9 were performed only in human RUES2-GLR ESCs before gastruloid induction; direct perturbation of candidate genes within differentiated gastruloid tissues at specific developmental stages would strengthen functional validation of the proteomic findings.

    The proteomic landscape and temporal dynamics of human and mouse gastruloid development · 2026 · DOI

  • Chemical perturbations in gastruloids were limited to MAPKAPK2 inhibition with MK2in1; systematic perturbation of other kinase signaling pathways implicated in gastrulation (e.g., MAPK, Wnt, Nodal pathways) with small-molecule inhibitors during human and mouse RA-gastruloid induction remains unexplored.

    The proteomic landscape and temporal dynamics of human and mouse gastruloid development · 2026 · DOI

  • The proteomic temporal dynamics of gastruloids were sampled only at discrete timepoints (72 and 144 h for mouse gastruloids; 24 and 120 h for human RA-gastruloids). High-resolution kinetic profiling at intermediate timepoints would be needed to capture the complete proteomic trajectory and identify transient protein expression states during gastruloid differentiation.

    The proteomic landscape and temporal dynamics of human and mouse gastruloid development · 2026 · DOI

  • The relationship between genome doubling events and zygote-like cancer stem cell formation through fusion mechanisms requires systematic investigation with whole genome sequencing datasets. Pan-cancer analysis of genome doubling patterns across different cancer types needs to be integrated with fusion-derived stem cell markers to establish causal links to cancer evolution and prognosis.

    On the potential origin of the zygote-like cancer stem cell with a focus on fusion for cell rescue · 2026 · DOI

  • The contribution of bone marrow-derived cells and macrophage-mediated pro-tumorigenic niche formation to zygote-like cancer stem cell maintenance in tissue-resident compartments has not been experimentally isolated from fusion events. Studies dissecting how nerve- and airway-associated interstitial macrophages regulate CD8 T cell immunity specifically in fusion-derived cancer populations are needed.

    On the potential origin of the zygote-like cancer stem cell with a focus on fusion for cell rescue · 2026 · DOI

  • The specific role of microRNAs (particularly miR-17-92 cluster and miR-34) in regulating somatic cell reprogramming and quiescence in fusion-derived cancer stem cells remains inadequately characterized. Direct mechanistic studies are needed to determine how these miRNAs modulate the transition between quiescent and proliferative states in zygote-like cancer stem cell populations.

    On the potential origin of the zygote-like cancer stem cell with a focus on fusion for cell rescue · 2026 · DOI

  • The temporal relationship between somatic mutations in tissue-resident cells and epigenetic changes in cancer initiation requires longitudinal investigation using multi-timepoint sampling prior to cancer diagnosis. The feasibility of detecting somatic mutations three years before clinical diagnosis to predict cancer emergence from fusion-derived stem cells needs empirical validation in prospective cohorts.

    On the potential origin of the zygote-like cancer stem cell with a focus on fusion for cell rescue · 2026 · DOI

  • The role of syncytin-mediated cell fusion in generating cancer stem cells with zygote-like characteristics has not been systematically compared across different cancer types and tissue origins. Comparative studies examining syncytin expression patterns and fusion efficiency in breast cancer versus other malignancies would clarify whether this mechanism is broadly applicable to the zygote-like cancer stem cell hypothesis.

    On the potential origin of the zygote-like cancer stem cell with a focus on fusion for cell rescue · 2026 · DOI

  • The mechanisms by which germline genes promote malignancy in cancer stem cells derived from zygote-like fusion events remain incompletely characterized. Specific investigation is needed into how massive expression of germ cell-specific genes in cancer cells relates to the zygote-like cancer stem cell origin hypothesis and fusion-based cell rescue mechanisms.

    On the potential origin of the zygote-like cancer stem cell with a focus on fusion for cell rescue · 2026 · DOI

  • (MDC), Berlin, Germany. 15Department of Pharmaceutical Sciences, College of Pharmacy, Robertson Life Sciences Building, Oregon State University, Portland, OR, USA. 16Department of Ophthalmology, Casey Eye Institute, Oregon Health and Science University, Portland, OR, USA. 17Department of Biomedical Engineering, Robertson Life Sciences Building, Oregon Health and Science University, Portland, OR, USA. 18Michael Smith Laboratories, School of Biomedical Engineering, University of British Columbia, Vancouver, BC, Canada. 19Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON, Canada. 20Institute Biomedical Engineering, University of Toronto, Toronto, ON, Canada. 21Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON, Canada. 22Department of Medical Sciences, Toronto, ON, Canada.

    Advancing regenerative medicine with RNA nanotechnology for chronic and end organ diseases · 2026 · DOI

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75 open questions have been extracted from the limitations and future-work passages of 306 Pluripotent Stem Cells Research papers in our library. Each one below links back to the study that raised it, so you can read the original claim in context.

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