Biology subjects

Izpisua Belmonte, J. C.

Publications and source records attributed to Izpisua Belmonte, J. C..

9 recordsLinked to original sources

bioRxiv2026

Visualizing the Epigenetic Landscape of Aging and Cellular Reprogramming: Optimized ATAC-see for Cells and Tissues

Spatial chromatin organization dictates cellular function and resilience, yet scalable imaging methods to quantify chromatin states in situ across aging and interventions are lacking. While ATAC-see can visualize accessible chromatin, its broader application is hindered by protocol variability, low throughput, and incompatibility with complex tissues. Here, we systematically optimize the ATAC-see workflow for robust, high-throughput quantitative imaging in fixed, adherent mammalian cells and fresh frozen tissues. We validate the platforms sensitivity to pharmacologic remodeling and apply it to replicative, chronological, and pathological aging in primary human fibroblasts, revealing progressive age-associated chromatin opening and heterochromatin remodeling. Furthermore, we demonstrate that our optimized ATAC-see captures rapid, reversible chromatin reorganization during OSK(M)-driven partial reprogramming of aged fibroblasts. Finally, we extend a cost-effective and accessible protocol to murine tissue sections, quantifying in situ age-dependent remodeling. This standardized framework establishes chromatin accessibility as a highly scalable, sequencing-compatible imaging biomarker for evaluating aging and rejuvenation. Summary StatementATAC-see was optimized for scalable, quantitative imaging of chromatin remodeling during aging and cellular reprogramming, and extended to characterize age-associated epigenetic changes across organs.

cell biology↗

bioRxiv2024

Optimization of an adeno-associated viral vector for keratinocytes in vitro and in vivo

BackgroundLocal gene therapies, including in vivo genome editing, are highly anticipated for the treatment of genetic diseases in skin, especially the epidermis. While the adeno-associated virus (AAV) is a potent vector for in vivo gene delivery, the lack of efficient gene delivery methods has limited its clinical applications. ObjectiveTo optimize the AAV gene delivery system with higher gene delivery efficiency and specificity for epidermis and keratinocytes (KCs), using AAV capsid and promoter engineering technologies. MethodsAAV variants with mutations in residues reported to be critical to determine the tropism of AAV2 for KCs were generated by site-directed mutagenesis of AAVDJ. The infection efficiency and specificity for KCs of these variants were compared with those of previously reported AAVs considered to be suitable for gene delivery to KCs in vitro and in vivo. Additionally, we generated an epidermis-specific promoter using the most recent short-core promoter and compared its specificity with existing promoters. ResultsA novel AAVDJ variant capsid termed AAVDJK2 was superior to the existing AAVs in terms of gene transduction efficiency and specificity for epidermis and KCs in vitro and in vivo. A novel tissue-specific promoter, termed the K14 SCP3 promoter, was superior to the existing promoters in terms of gene transduction efficiency and specificity for KCs. ConclusionThe combination of the AAVDJK2 capsid and K14 SCP3 promoter improves gene delivery to epidermis in vivo and KCs in vitro. The novel AAV system may benefit experimental research and development of new epidermis-targeted gene therapies.

bioengineering↗

bioRxiv2023

Efficient workflow for validating homology-independent targeted integration-mediated gene insertion in rod photoreceptor cells to treat dominant-negative mutations causing retinitis pigmentosa

Among the genome-editing methods for repairing disease-causing mutations resulting in dominant inhibition, homology-independent targeted integration (HITI)-mediated gene insertion of the normal form of the causative gene is useful because it allows the development of mutation-agnostic therapeutic products. For the rapid optimization and validation of highly effective HITI-treatment gene constructs against dominant-negative inheritance of inherited retinal dystrophy, we improved the gene constructs available in both plasmid and adeno-associated virus (AAV) vectors, and established a workflow that uses in vivo electroporation to verify the in vivo efficacy. By targeting the mouse Rhodopsin gene, we derived a construct in which HITI-mediated gene insertion occurs in 80%-90% of transduced mouse rod photoreceptor cells. This construct suppressed degeneration and induced visual restoration in the mutant mice. The HITI-treatment constructs for the rhodopsin gene were shown to be effective in AAV vectors, and this construction is available for the mouse Peripherin 2 gene. These findings suggest that the workflow reported here may be useful for the generation of HITI-treatment constructs for various target genes and for the development of gene therapy products.

molecular biology↗

bioRxiv2023

Convergence of aging- and rejuvenation-related epigenetic alterations on PRC2 targets

Rejuvenation of tissues in physiologically aging mice can be accomplished by long-term partial reprogramming via expression of reprogramming factors (Oct4, Sox2, Klf4 and c-Myc). To investigate the epigenetic determinants of partial reprogramming-mediated rejuvenation, we used whole genome bisulfite sequencing to carry out unbiased comprehensive profiling of DNA methylation changes in skin from mice subjected to partial reprogramming, as well as young and untreated old controls. We found a striking convergence of age- and rejuvenation-related epigenetic alterations on targets of the Polycomb repressive complex 2 (PRC2). These results are also supported by a likewise prominent enrichment of PRC2 targets in gene expression data, suggesting that PRC2 activity can modulate aging and mediate tissue rejuvenation.

genomics↗

bioRxiv2023

In vivo reprogramming of wound-resident cells generates skin with hair

Summary ParagraphMammalian skin appendages, such as hair follicles and sweat glands, are complex mini-organs formed during skin development1, 2. As wounds heal, the resulting scar tissue lacks skin appendages. The clinical regeneration of skin appendages is an ongoing challenge3, 4. Skin epithelial tissues have been regenerated in vivo by cellular reprogramming5, 6, but the de novo generation of skin appendages has not previously been achieved. Here, we show that transplantation of a type of epithelial cell and two types of mesenchymal cells, reprogrammed from adult mouse subcutaneous mesenchymal cells to mimic developing skin cells, resulted in the generation of skin-appendage-like structures. Furthermore, with the development of a new AAV serotype, in vivo reprogramming of wound-resident cells with the same reprogramming factors generates skin with de novo appendages in adult mice. These findings may provide new therapeutic avenues for skin regeneration and frequent aging-associated skin appendage disorders, such as hair loss and dry skin, and may extend to other tissues and organs. This study also provides the potential for de novo generation of complex organs in vivo.

cell biology↗

bioRxiv2020

Single-cell Individual Complete mtDNA Sequencing Uncovers Hidden Mitochondrial Heterogeneity in Human and Mouse Oocytes

The ontogeny and dynamics of mtDNA heteroplasmy remain unclear due to limitations of current mtDNA sequencing methods. We developed individual Mitochondrial Genome sequencing (iMiGseq) of full-length mtDNA for ultra-sensitive variant detection, complete haplotyping, and unbiased evaluation of heteroplasmy levels, all at the individual mtDNA molecule level. iMiGseq uncovers unappreciated levels of heteroplasmic variants in single healthy human oocytes well below the current 1% detection limit, of which numerous variants are detrimental and could contribute to late-onset mitochondrial disease and cancer. Extreme mtDNA heterogeneity among oocytes of the same mouse female, and a strong selection against deleterious mutations in human oocytes are observed. iMiGseq could comprehensively characterize and haplotype single-nucleotide and structural variants of mtDNA and their genetic linkage in NARP/Leigh syndrome patient-derived cells. Therefore, iMiGseq could not only elucidate the mitochondrial etiology of diseases, but also help diagnose and prevent mitochondrial diseases with unprecedented precision.

genomics↗

bioRxiv2020

Derivation and characterization of chimera-competent eXtra-Embryonic eNdoderm 1 (XEN) cells from pig blastocysts

In this article, we report for the first time the derivation and characterization of extra-embryonic endoderm (XEN) cells from primitive endoderm (PrE) of porcine (p) embryos. The pXEN cells can be reliably and reproducibly generated from parthenote, in vitro and in vivo derived embryos. The pXEN cells retained all the hallmarks of PrE including expression of canonical PrE and XEN cell markers (GATA4, GATA6, SOX17, SALL4, FOXA2, and HNF4A). Transcriptome analysis further confirmed their XEN cell origin. The pXEN cells when introduced into blastocyst stage embryo contributed to wide-spread chimerism including visceral yolk sac, chorion, as well as embryonic gut and liver primordium in the fetus. The pXEN cells were shown to be an efficient nuclear donor for generating cloned offspring. Taken together, pXEN cells fulfil a longstanding need for a stable, chimera-competent, and nuclear transfer-compatible porcine embryonic cells with applications for agriculture and medicine. Significance StatementWe report for the first time, the derivation and characterization of extraembryonic endoderm (XEN) stem cells from porcine (p) embryos. The pXEN cells can be reliably and reproducibly derived from primitive endoderm precursors. When injected into blastocyst-stage embryos, the pXEN cells have contributed to wide-spread chimerism including visceral yolk sac, chorion of the extraembryonic membranes, as well as definitive endoderm of the fetus, primarily the embryonic gut and liver primordium. Additionally, these XEN cells have proven to be an efficient nuclear donor for generating cloned offspring. These newly discovered stem cells provide a novel model for studying lineage segregation, as well as a source for interspecies chimeras for generating endodermal organs, and for genome editing in livestock.

developmental biology↗

bioRxiv2019

A novel microRNA-based strategy to expand the differentiation potency of stem cells

Full differentiation potential along with self-renewal capacity is a major property of pluripotent stem cells (PSCs). However, the differentiation capacity frequently decreases during expansion of PSCs in vitro. We show here that transient exposure to a single microRNA, expressed at early stages during normal development, improves the differentiation capacity of already-established murine and human PSCs. Short exposure to miR-203 in PSCs (miPSCs) results in expanded differentiation potency as well as improved efficiency in stringent assays such as tetraploid complementation and human-mouse interspecies chimerism. Mechanistically, these effects are mediated by direct repression of de novo DNA methyltransferases Dnmt3a and Dnmt3b, leading to transient and reversible erasing of DNA methylation. As a proof of concept, miR-203 improves differentiation and maturation of PSCs into cardiomyocytes in vitro as well as cardiac regeneration in vivo, after cardiac injury. These data support the use of transient exposure to miR-203 as a general and single method to reset the epigenetic memory in PSCs, and improve their use in regenerative medicine.

cell biology↗

bioRxiv2019

GRP78 promotes stemness in normal and neoplastic cells

Reliable approaches to identify stem cell mechanisms that mediate aggressive cancer could have great therapeutic value, based on the growing evidence of embryonic signatures in metastatic cancers. However, how to best identify and target stem-like mechanisms aberrantly acquired by cancer cells has been challenging. We harnessed the power of reprogramming to examine GRP78, a chaperone protein generally restricted to the endoplasmic reticulum in normal tissues, but which is expressed on the cell surface of human embryonic stem cells and many cancer types. We have discovered that (1) cell surface GRP78 (sGRP78) is expressed on iPSCs and is important in reprogramming, (2) sGRP78 promotes cellular functions in both pluripotent and breast cancer cells (3) overexpression of GRP78 in breast cancer cells leads to an induction of a CD24-/CD44+ tumor initiating cell (TIC) population (4) sGRP78+ breast cancer cells are enriched for stemness genes and appear to be a subset of TICs (5) sGRP78+ breast cancer cells show an enhanced ability to seed metastatic organ sites in vivo. These collective findings show that GRP78 has important functions in regulating both pluripotency and oncogenesis, and suggest that sGRP78 marks a stem-like population in breast cancer cells that has increased metastatic potential in vivo.

cancer biology↗